Methine-Substituted Cyanine Dye Compounds

ABSTRACT

Cyanine dye compounds having a substituted methine moiety that are nucleic acid stains, particularly for fluorescent staining of RNA, including compounds having the formula 
     
       
         
         
             
             
         
       
     
     where R 1  is a C 1 -C 6  alkyl, sulfoalkyl, carboxyalkyl or C 1 -C 6  alkoxy; each R 2  is independently selected from the group consisting of H, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, fused benzo, trifluoromethyl, amino, sulfo, carboxy and halogen, that is optionally further substituted; at least one of R 3 , R 4 , and R 5  is an alkyl, aryl, heteroaryl, cyclic, or heterocyclic moiety that is optionally substituted by alkyl, amino, aminoalkyl, carboxy, nitro, or halogen; and the remaining R 3 , R 4  or R 5  are hydrogen; X is S, O, or Se; and D is a substituted or unsubstituted pyridinium, quinolinium or benzazolium moiety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. Ser. No. 14/662,397, filed Mar.19, 2015, which is a continuation of U.S. Ser. No. 13/902,076, filed May24, 2013 (now U.S. Pat. No. 9,040,561), which is a continuation of U.S.Ser. No. 12/851,030, filed Aug. 5, 2010 (now U.S. Pat. No. 8,470,529),which is a continuation of U.S. Ser. No. 11/005,861, filed Dec. 6, 2004(now U.S. Pat. No. 7,776,529), which claims priority to U.S. Ser. No.60/527,142, filed Dec. 5, 2003, and U.S. Ser. No. 60/554,452, filed Mar.18, 2004, which disclosures are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to cyanine compounds useful for stainingnucleic acids, including RNA. The invention has applications in thefields of molecular biology, particularly with respect tofluorescence-based assays.

BACKGROUND OF THE INVENTION

In selected fields of life sciences research, including for examplebiological, biomedical, genetic, fermentation, aquaculture,agricultural, forensic and environmental research, there may often occurthe need to identify nucleic acids, qualitatively and quantitatively, inpure solutions and in biological samples. Such applications may benefitfrom fast, sensitive, and selective methodologies for detecting and/orquantifying nucleic acids of interest.

In particular, it may be helpful in some research venues to providemolecular species that at least somewhat selectively stain RNA even inthe presence of DNA. That is, the probe or reagent may permit theresearcher to distinguish RNA present in a sample from DNA in the samesample.

SUMMARY

Embodiments of the present invention provide nucleic acid reportercompounds, which are cyanine dyes that comprise at substituted methinebridge. These reporter compounds find use as nucleic acid stains,particularly for the fluorescent detection/quantitation of DNA.

In one embodiment, the nucleic acid reporter molecules have the formula:

wherein at least one of R³, R⁴, and R⁵ is an alkyl, substituted alkyl, a5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, asubstituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or7-membered aryl or a substituted 5-, 6- or 7-membered aryl; and theremaining R³, R⁴ or R⁵ are hydrogen.

In an exemplary embodiment at least one of R³, R⁴, and R⁵ is asubstituted alkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl,a substituted 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or7-membered heteroaryl, or a substituted 5-, 6- or 7-membered aryl thatis substituted by an alkyl, —(CH₂)_(k)—NR⁶R⁷; —COOR⁸, NO₂, or halogen,wherein k is an integer from 0 to about 6. The substituents R⁶, R⁷ andR⁸ are independently hydrogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, sulfoalkyl or aminoalkyl. In one aspect at least oneof R³ and R⁴ is a thiophenyl, substituted thiophenyl, adamantyl,substituted adamantly, phenyl, substituted phenyl, alkyl, substitutedalkyl, benzyl or substituted benzyl.

These nucleic acid reporter molecules exhibit a fluorescence enhancementwhen non-covalently associated with a nucleic acid molecule. In oneaspect, the fluorescence enhancement is greater when the nucleic acid isRNA than when the nucleic acid is DNA. In another aspect, thefluorescence enhancement is greater when the nucleic acid is DNA thanwhen the nucleic acid is RNA.

The R¹ substituent is independently hydrogen, carboxy, sulfo, phosphate,phosphonate, amino, hydroxyl, trifluoromethyl, halogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, alkylamino, substitutedalkylamino, dialkylamino, substituted dialkylamino, aminoalkyl,substituted aminoalkyl, fused benzene, substituted fused benzene,reactive group, solid support or carrier molecule and t is an integerfrom 1 to 4. In one aspect R¹ is alkoxy or halogen. In another aspect R¹is methoxy, Br, Cl, or F.

The R² substituent is an alkyl, substituted alkyl, arylalkyl,substituted arylalkyl, heteroalkyl, substituted heteroalkyl, alkoxy,substituted alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo,sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, ortrialkylammonium. In one aspect R² is methyl, ethyl, propyl, or—(CH₂)₃SO₃ ⁻.

X is O, S or Se.

The D is a substituted pyridinium, unsubstituted pyridinium, substitutedquinolinium, unsubstituted quinolinium, substituted benzazolium orunsubstituted benzazolium moiety. In an exemplary embodiment, D has theformula:

wherein R¹¹ is hydrogen, substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl,unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl,substituted heteroarylalkyl; unsubstituted heteroarylalkyl, substitutedheteroaryl, unsubstituted heteroaryl substituted cycloalkyl,unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstitutedheterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstitutedalkylamino, substituted alkylthio, unsubstituted alkylthio, reactivegroup, solid support, or carrier molecule and R¹² is an alkyl,substituted alkyl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, alkoxy, substituted alkoxy, carboxy,carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino,aminoalkyl, alkylamino, dialkylamino, or trialkylammonium.Alternatively, R¹¹ in combination with an adjacent R¹¹ or R¹², togetherwith the atoms to which they are joined, form a ring which is a 5-, 6-or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-memberedheterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, a substituted 5-,6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heteroaryl, asubstituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or 7-membered arylor a substituted 5-, 6- or 7-membered aryl.

In an exemplary embodiment of the kits, the nucleic acid reportermolecule has the formula:

-   -   or the formula

Additional embodiments of the present invention provide methods ofdetecting the presence or absence of nucleic acid, including method fordetecting the presence or absence of RNA in the presence of DNA. Thepresent methods comprise:

-   -   a. combining a present nucleic acid reporter molecule with the        sample to prepare a labeling mixture;    -   b. incubating the labeling mixture for a sufficient amount of        time for the nucleic acid reporter molecule to associate with        nucleic acid in the sample to form an incubated mixture;    -   c. illuminating the incubated sample with an appropriate        wavelength to form an illuminated mixture; and,    -   d. observing the illuminated mixture whereby the presence or        absence of the nucleic acid in a sample is detected.

In one aspect, the nucleic acid reporter molecule has a RNA/DNA ratio offluorescence enhancement greater than about one.

Also provided is a staining solution comprising a present nucleic acidreporter molecule and a detergent. The detergent is typically present inan aqueous solution at a concentration from about 0.0001% to about0.05%. Detergents include CHAPTS, Triton-X, SDS and Tween 20. In afurther aspect, the staining solution contains a low concentration ofDNA, a low concentration being about 0.1 μg/ml to about 0.5 μg/ml.

Further embodiments provide complexes of the present compoundsnon-covalently associated with nucleic acid and compositions comprisinga present compound and a sample. In one aspect the sample comprisesbiological fluids, buffer solutions, live cells, fixed cells, eukaryoticcells, prokaryotic cells, nucleic acid polymers, nucleotides,nucleosides, a polymeric gel or tissue sections. In a further aspect thesample is present in an aqueous solution, in or on a microarray or amicrowell plate.

Additional embodiments of the present invention provide kits for thedetection of nucleic acid, wherein the kit comprises any compound of thepresent invention. In a further embodiment, the kits compriseinstructions for the detection of nucleic acid, particularlyinstructions for the detection of RNA in the presence of DNA. In yetanother further embodiment, the kits comprises at least one componentthat is a sample preparation reagent, a buffering agent, an organicsolvent, an aqueous nucleic acid reporter molecule dilution buffer,nucleic acid control, or an additional detection reagent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A plot showing the intensity and emission spectra of thefluorescent signal from Compound 20 when associated with either rRNA orDNA (calf thymus) in solution, with excitation at 630 nm, as describedin Example 7. Compound 20 demonstrates a 12 to 13-fold increase insignal when bound to RNA when compared to DNA.

FIG. 2: A plot showing the intensity of the fluorescent signal fromCompound 20 in the presence of rRNA, DNA (calf thymus) and a mixture ofRNA and DNA in solution. The results indicate that Compound 20, insolution, demonstrates selectivity for rRNA in the presence of 1000ng/ml of DNA, as described in Example 9.

FIG. 3: A plot showing the fluorescence intensity of Compound 20 whenassociated with rRNA, tRNA and mRNA, respectively, in increasingconcentrations of the RNA. Compound 20 demonstrates an equal affinityfor the three different species of RNA, as described in Example 8.

FIG. 4: A plot showing the intensity of the fluorescent signal fromCompound 20 at 0.025 μM when bound to varying concentrations of rRNA, ora 1:1 mixture of RNA and DNA in solution. FIG. 4 is an overlay of twographs with concentration of RNA 0-200 ng/mL, as described in Example10.

FIG. 4A: A plot showing the linear correlations of the plots of FIG. 4.The similarity of the relationship between fluorescence emissionintensity and RNA concentration, even in the presence of varying amountsof DNA, show the selectivity of Compound 20 for RNA, as described inExample 10.

DETAILED DESCRIPTION Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a compound” includes aplurality of compounds and reference to “a nucleic acid” includes aplurality of nucleic acids and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for the purposes of understanding the present disclosure.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds described herein may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds disclosed herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are intended to beencompassed within the scope of the present invention.

Where a disclosed compound includes a conjugated ring system, resonancestabilization may permit a formal electronic charge to be distributedover the entire molecule. While a particular charge may be depicted aslocalized on a particular ring system, or a particular heteroatom, it iscommonly understood that a comparable resonance structure can be drawnin which the charge may be formally localized on an alternative portionof the compound.

Selected compounds having a formal electronic charge may be shownwithout an appropriate biologically compatible counterion. Such acounterion serves to balance the positive or negative charge present onthe compound. As used herein, a substance that is biologicallycompatible is not toxic as used, and does not have a substantiallydeleterious effect on biomolecules. Examples of negatively chargedcounterions include, among others, chloride, bromide, iodide, sulfate,alkanesulfonate, arylsulfonate, phosphate, perchlorate,tetrafluoroborate, tetraarylboride, nitrate and anions of aromatic oraliphatic carboxylic acids. Preferred counterions may include chloride,iodide, perchlorate and various sulfonates. Examples of positivelycharged counterions include, among others, alkali metal, or alkalineearth metal ions, ammonium, or alkylammonium ions.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH moietytherefrom.

The term “affinity” as used herein refers to the strength of the bindinginteraction of two molecules, such as a nucleic acid polymer and anintercalating agent or a positively charged moiety and a negativelycharged moiety.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present invention contain betweenabout one and about twenty five carbon atoms (e.g. methyl, ethyl and thelike). Straight, branched or cyclic hydrocarbon chains having eight orfewer carbon atoms will also be referred to herein as “lower alkyl”. Inaddition, the term “alkyl” as used herein further includes one or moresubstitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The term “amino” or “amine group” refers to the group —NR′R″ (or NRR′R″)where R, R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine being an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —NRR′R″ and itsbiologically compatible anionic counterions.

The term “aryl” as used herein refers to cyclic aromatic carbon chainhaving twenty or fewer carbon atoms, e.g., phenyl, naphthyl, biphenyl,and anthracenyl. One or more carbon atoms of the aryl group may also besubstituted with, e.g., alkyl; aryl; heteroaryl; a halogen; nitro;cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl-, orarylthio; amino, alkylamino, arylamino, dialkyl-, diaryl-, orarylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsufonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two ormore alkyl or heteroalkyl substituents of an aryl group may be combinedto form fused aryl-alkyl or aryl-heteroalkyl ring systems (e.g.,tetrahydronaphthyl). Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P, Se, and S, and wherein thenitrogen, phosphorous and sulfur atoms are optionally oxidized, and thenitrogen heteroatom is optionally be quaternized. The heteroatom(s) 0,N, P, S, Se, and Si may be placed at any interior position of theheteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or aspart of another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms selected from N, O, S, and Se wherein the nitrogenand sulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound includes morethan one R group, for example, each of the R groups is independentlyselected as are each R′, R″, R′ and R″″ groups when more than one ofthese groups is present. When R′ and R″ are attached to the samenitrogen atom, they can be combined with the nitrogen atom to form a 5-,6-, or 7-membered ring. For example, —NR′R″ is meant to include, but notbe limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄) alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′ and R″″ groupswhen more than one of these groups is present. In the schemes thatfollow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″F″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P), selenium (Se), and silicon (Si), amongothers.

The term “amino” or “amine group” refers to the group —NR′R″ (orN+RR′R″) where R, R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl. A substituted amine being an amine group wherein R′ or R″ isother than hydrogen. In a primary amino group, both R′ and R″ arehydrogen, whereas in a secondary amino group, either, but not both, R′or R″ is hydrogen. In addition, the terms “amine” and “amino” caninclude protonated and quaternized versions of nitrogen, comprising thegroup —N+RR′R″ and its biologically compatible anionic counterions.

The term “attachment site” as used herein refers to a site on a moietyor a molecule, e.g. a quencher, or a fluorescent dye, to which iscovalently attached, or capable of being covalently attached, to alinker or another moiety.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “Carboxyalkyl” as used herein refers to a group having thegeneral formula —(CH₂)_(n)COOH wherein n is 1-18.

The term “carrier molecule” as used herein refers to a biological or anon-biological component that is covalently bonded to a compound of thepresent invention. Such components include, but are not limited to, anamino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof.

The term “complex” as used herein refers to the association of two ormore molecules, usually by non-covalent bonding.

The term “cyanine dye” as used herein refers to a fluorogenic compoundthat comprises 1) a substituted or unsubstituted benzazolium moiety, 2)a polymethine bridge and 3) a substituted or unsubstituted benzazolium,pyridinium or quinolinium moiety. These monomer or dye moieties arecapable of forming a non-covalent complex with nucleic acids anddemonstrating an increased fluorescent signal after formation of thenucleic acid-dye complex.

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of the above parameters.

The term “kit” as used refers to a packaged set of related components,typically one or more compounds or compositions.

The term “Linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-20 nonhydrogenatoms selected from the group consisting of C, N, O, S and P thatcovalently attach the fluorogenic or fluorescent compounds to anothermoiety such as a chemically reactive group or a biological andnon-biological component. Exemplary linking members include a moietythat includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like. A“cleavable linker” is a linker that has one or more cleavable groupsthat may be broken by the result of a reaction or condition. The term“cleavable group” refers to a moiety that allows for release of aportion, e.g., a fluorogenic or fluorescent moiety, of a conjugate fromthe remainder of the conjugate by cleaving a bond linking the releasedmoiety to the remainder of the conjugate. Such cleavage is eitherchemical in nature, or enzymatically mediated. Exemplary enzymaticallycleavable groups include natural amino acids or peptide sequences thatend with a natural amino acid.

In addition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases,hydroxylamine, light (e.g., nitrobenzyl derivatives, phenacyl groups,benzoin esters), and heat. Many cleavable groups are known in the art.See, for example, Jung et al., Biochim. Biophys. Acta, 761: 152-162(1983); Joshi et al., J. Biol. Chem., 265: 14518-14525 (1990); Zarlinget al., J. Immunol., 124: 913-920 (1980); Bouizar et al., Eur. J.Biochem., 155: 141-147 (1986); Park et al., J. Biol. Chem., 261: 205-210(1986); Browning et al., J. Immunol., 143: 1859-1867 (1989). Moreover, abroad range of cleavable, bifunctional (both homo- andhetero-bifunctional) spacer arms are commercially available.

An exemplary cleavable group, an ester, is cleavable group that may becleaved by a reagent, e.g. sodium hydroxide or hydroxylamine, resultingin a carboxylate-containing fragment and a hydroxyl-containing product.

The linker can be used to attach the compound to another component of aconjugate, such as a targeting moiety (e.g., antibody, ligand,non-covalent protein-binding group, etc.), an analyte, a biomolecule, adrug and the like.

As used herein, “nucleic acid” or “nucleic acid polymer” means DNA, RNA,single-stranded, double-stranded, or more highly aggregatedhybridization motifs, and any chemical modifications thereof.Modifications include, but are not limited to, those providing chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, and electrostatic interaction to the nucleic acid ligand basesor to the nucleic acid ligand as a whole. Such modifications include,but are not limited to, peptide nucleic acids (PNAs), phosphodiestergroup modifications (e.g., phosphorothioates, methylphosphonates),2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases, isocytidine and isoguanidine and the like. Nucleicacids can also include non-natural bases, such as, for example,nitroindole. Modifications can also include 3′ and 5′ modifications suchas capping with a quencher, a fluorophore, a reactive group or anothermoiety.

The term “nucleic acid reporter molecule” as used herein refers to thepresent cyanine compounds wherein the methine bridge is substituted byat least one aryl, heteroaryl or substituted alkyl group.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Thereactive group is a moiety, such as carboxylic acid, amine, alcohol orsuccinimidyl ester, on the compounds of the present invention that iscapable of chemically reacting with a functional group on a differentcompound to form a covalent linkage resulting in a fluorescent orfluorogenic labeled component. Reactive groups generally includenucleophiles, electrophiles and photoactivatable groups.

Exemplary reactive groups include, but not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, thiosulfates, sulfenic acidsisonitriles, amidines, imides, imidates, nitrones, hydroxylamines,oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters,sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides,carbodiimides, carbamates, imines, azides, azo compounds, azoxycompounds, and nitroso compounds. Reactive functional groups alsoinclude those used to prepare bioconjugates, e.g., N-hydroxysuccinimideesters, maleimides and the like. Methods to prepare each of thesefunctional groups are well known in the art and their application to ormodification for a particular purpose is within the ability of one ofskill in the art (see, for example, Sandler and Karo, eds. ORGANICFUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989).

The term “reporter molecule” as used herein refers to any luminescentmolecule that is capable of associating with a nucleic acid polymer andproducing a detectable signal. Typically, reporter molecules includeunsymmetrical cyanine dyes, homo- or heterodimers or oligomers ofcyanine dyes, ethidium bromide, DAPI, Hoechst, acridine and styryl dyesthat are capable of producing a detectable signal upon appropriatewavelength excitation.

The term “salt thereof,” as used herein includes salts of the agents ofthe invention and their conjugates, which are preferably prepared withrelatively nontoxic acids or bases, depending on the particularsubstituents found on the compounds described herein. When compounds ofthe present invention contain relatively acidic functionalities, baseaddition salts can be obtained by contacting the neutral form of suchcompounds with a sufficient amount of the desired base, either neat orin a suitable inert solvent. Examples of base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofaddition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulf uric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

The term “sample” as used herein refers to any material that may containnucleic acid. The sample may also include diluents, buffers, detergents,and contaminating species, debris and the like that are found mixed withthe target. Illustrative examples include urine, sera, blood plasma,total blood, saliva, tear fluid, cerebrospinal fluid, secretory fluidsand the like. Also included are solid, gel or substances such as mucus,body tissues, cells and the like suspended or dissolved in liquidmaterials such as buffers, extractants, solvents and the like.Typically, the sample is a live cell, a biological fluid that comprisesendogenous host cell proteins, nucleic acid polymers, nucleotides,oligonucleotides, peptides and buffer solutions. The sample may bedissolved or suspended in an aqueous solution, a viable cell culture orimmobilized on a solid or semi solid surface such as a polyacrylamidegel, membrane blot or on a microarray.

The term “solid support,” as used herein, refers to a material that issubstantially insoluble in a selected solvent system, or which can bereadily separated (e.g., by precipitation) from a selected solventsystem in which it is soluble. Solid supports useful in practicing thepresent invention can include groups that are activated or capable ofactivation to allow selected species to be bound to the solid support.Solid supports may be present in a variety of forms, including a chip,wafer or well, onto which an individual, or more than one compound, ofthe invention is bound such as a polymeric bead or particle.

The term “sulfoalkyl,” as used herein refers to a group having thegeneral formula —(CH₂)_(n)SO⁻³ wherein n is 1-18.

The Compounds

In general, for ease of understanding the present invention, the nucleicacid reporter molecules and corresponding substituents will first bedescribed in detail, followed by the many and varied methods in whichthe compounds find uses, which is followed by exemplified methods of useand synthesis of novel compounds that are particularly advantageous foruse with the methods of the present invention.

The compounds of the present disclosure typically exhibit a fluorescenceenhancement when non-covalently associated with a nucleic acid. Forselected compounds, the fluorescence enhancement is greater when thenucleic acid is RNA than when the nucleic acid is DNA.

In one embodiment the present invention provides nucleic acid complexingcompounds that comprise at substituted methine bridge. Without wishingto be bound by theory, it is believed that the selection of anonhydrogen methine substituent (one of R³, R⁴, and R⁵) may permit thetuning of selectivity and affinity of the resulting compound forparticular nucleic acids. In particular, it is believed that thepresence of a nonhydrogen methine substituent increases the selectivityand affinity of the resulting compound for RNA, when compared to thesame compound when binding to DNA. The fluorescence enhancement of theresulting RNA complex may be greater than that of the corresponding DNAcomplex.

The methine substituents is typically bulky such that is affects theassociation with DNA. Typically such substituents include, but are notlimited to, alkyl, substituted alkyl, a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-memberedcycloalkyl, a 5-, 6- or 7-membered heteroaryl, a substituted 5-, 6- or7-membered heteroaryl, a 5-, 6- or 7-membered aryl or a substituted 5-,6- or 7-membered aryl.

Typically, the nucleic acid complexing compounds are unsymmetricalcyanine dyes including, but are not limited to, any compound disclosedin U.S. Pat. Nos. 4,957,870; 4,883,867; 5,436,134; 5,658,751, 5,534,416and 5,863,753, when substituted with a negatively charged moiety. Thereis no intended limitation on the nucleic acid complexing compound.

In an exemplary embodiment, the present nucleic acid reporter moleculesmay be described by the formula:

wherein at least one of R³, R⁴, and R⁵ is an alkyl, substituted alkyl, a5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, asubstituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or7-membered aryl or a substituted 5-, 6- or 7-membered aryl; and theremaining R³, R⁴ or R⁵ are hydrogen.

Each nonhydrogen methine substituent may itself be further substitutedone or more times by alkyl, amino, carboxy, nitro, or halogen. Typicallyone or two of R³, R⁴, and R⁵ is nonhydrogen. More typically, exactly oneof R³, R⁴, and R⁵ is nonhydrogen. In one aspect of the invention, R⁴ isnonhydrogen.

In particular, where at least one of R³, R⁴, and R⁵ is an alkyl, aryl,heteroaryl, cyclic, or heterocyclic moiety, the moiety may include 3-20non-hydrogen atoms selected from the group consisting of O, N, S, P andC, and be optionally further substituted by C₁-C₆ alkyl, —NR⁶R⁷; —COOR⁸,NO₂, or halogen, wherein R⁶, R⁷, and R⁸ are independently hydrogen,C₁-C₆ alkyl, C₁-C₆ alkoxy, sulfoalkyl or aminoalkyl.

Where R⁴ is a nonhydrogen substituent, R⁴ may be alkyl, cycloalkyl,heteroaryl, or aryl. More particularly, R⁴ may be selected from linearor branched alkyl, cycloalkyl, thiophenyl, furanyl, and phenylsubstituents, that may be further substituted by one or more ofhydrogen, F, Cl, CF₃, —CO₂CH₃, NO₂, amino, C₁-C₆ alkoxy, phenoxy, andC₁-C₆ alkyl that is itself optionally further substituted by amino,sulfo, or carboxy.

The R¹ substituents may include any aryl group substituent, includingadditional fused 5- or 6-membered rings. Where each R¹ is independentlyhydrogen, carboxy, sulfo, phosphate, phosphonate, amino, hydroxyl,trifluoromethyl, halogen, alkyl, substituted alkyl, alkoxy, alkylamino,substituted alkylamino, dialkylamino, substituted dialkylamino,aminoalkyl, substituted aminoalkyl, fused benzene, substituted fusedbenzene, reactive group, solid support or carrier molecule, wherein t isan integer from 1 to 4. Each alkyl portion of which is optionallysubstituted by alkyl group substituents, as described above. Inparticular, the alkyl groups substituents may be selected from the groupconsisting of carboxy, sulfo, phosphate, phosphonate, amino, andhydroxy. Where R¹ is nonhydrogen, R¹ may be an alkoxy substituent, or ahalogen substituent, more preferably at least one R¹ is methoxy, Br, Cl,or F.

The R² substituent is an alkyl, substituted alkyl, arylalkyl,substituted arylalkyl, heteroalkyl, substituted heteroalkyl, alkoxy,substituted alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo,sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, ortrialkylammonium. In particular, R² may be selected from lower alkyl orsulfoalkyl, more preferably methyl, ethyl, propyl, or —(CH₂)₃SO₃ ⁻.

The X moiety is selected from S, O, or Se, forming a benzothiazole,benzoxazole, or benzoselenazole heterocyclic ring system, respectively.Typically, X is S or O, and more typically, X is S.

The D moiety is a substituted or unsubstituted ring system, includingpyridinium, quinolinium or benzazolium ring systems. For example, the Dmoiety may include the following ring systems (additional substituentsomitted for clarity):

The D ring system is optionally further substituted by any aryl groupsubstituent, as described above.

In an exemplary embodiment, the D moiety has the formula:

wherein R¹¹ is hydrogen, substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl,unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl,substituted heteroarylalkyl; unsubstituted heteroarylalkyl, substitutedheteroaryl, unsubstituted heteroaryl, substituted cycloalkyl,unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstitutedheterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstitutedalkylamino, substituted alkylthio, unsubstituted alkylthio, reactivegroup, solid support, or carrier molecule

The R¹² substituent is an alkyl, substituted alkyl, arylalkyl,substituted arylalkyl, heteroalkyl, substituted heteroalkyl, alkoxy,substituted alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo,sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, ortrialkylammonium; or R¹¹ in combination with an adjacent R¹¹ or R¹²,together with the atoms to which they are joined, form a ring which is a5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, asubstituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or7-membered aryl or a substituted 5-, 6- or 7-membered aryl.

In an exemplary embodiment, the present compounds have the formula:

-   -   or the formula

The compounds disclosed herein are readily modified to permit selectablealteration of the permeability, affinity, absorption, and emissionproperties (for example, see U.S. Pat. No. 5,658,751, herebyincorporated by reference). The resulting compounds may be tailored tocover most of the visible and near-infrared spectrum.

Synthesis

The reporter compounds disclosed herein may be prepared using a two-partsynthetic strategy: First, the preparation of an benzazolium moietycontaining a methine substituent, followed by addition of theappropriate pyridinium, quinolinium or benzazolium moiety. Typicallyeach component is selected so as to incorporate the desired and/orappropriate chemical substituents, or functional groups that may beconverted to the desired and/or appropriate chemical substituents. Thesynthetic strategies and procedures that may be used to prepare andcombine these precursors so as to yield the disclosed compounds isgenerally well understood by one skilled in the art, including a varietyof modifications and variations thereof. Herein are some useful generalmethods for the synthesis of selected compounds, including theincorporation of some chemical modifications.

Synthesis of the disclosed compounds, including the desired substituenton the trimethine bridge, may be facilitated by the initial preparationof selected intermediate compounds. For example, the intermediatebenzazole moiety may be described using one of the following generalstructures. For the sake of simplicity, most of the substituents areshown as hydrogen, however this should not be considered limiting thebreadth of the following disclosure.

If X is O, the precursor compound is a benzoxazolium; if X is S it is abenzothiazolium; if X is Se it is a benzoselenazolium. The commercialavailability of suitable starting materials and relative ease ofsynthesis may make compounds where X is O or S more attractive.

The desired R¹ substituents are typically incorporated in the parentbenzazole molecule prior to quaternization with an alkylating agent. R²is typically incorporated by quaternization of the parent heterocyclewith an alkylating agent, typically a source of (R²)⁺. The alkylatingreagent may be an alkyl halide such as ethyl iodide, an alkylsulfonatesuch as methyl p-toluenesulfonate or a cyclic sulfonate such aspropanesultone or butanesultone. The benzazolium moiety may then besubsequently reacted with an aromatic acid chloride to generate thedesired benzoketone key intermediate which can be activated byphosphorous oxychloride followed by the addition of the desiredpyridinium, quinolinium or benzazolium moiety, or “D” moiety.

Although the above reaction scheme depicts the use of a benzoyl chloridereagent, it should be appreciated that the selection of reagent isdependent upon the nature of the methine substituent desired, and that avariety of reagents may be used, as shown in Example 1.

Similarly, although the D moiety depicted above is a benzazole, avariety of D intermediates, including pyridinium, quinolinium andbenzazolium intermediates, may be used to complete the synthesis asdescribed above. Selected examples of such variations are described inExamples 1-6.

Specific examples of D moieties appropriate for the above syntheticscheme may be found in, for example, U.S. Pat. No. 5,436,134, herebyincorporated by reference. The pyridinium, quinolinium or benzazoliummoiety may be fused to additional rings, resulting in dyes that absorband emit at longer wavelengths (for example, see U.S. Pat. No.6,027,709, hereby incorporated by reference).

Reactive Groups, Carrier Molecules and Solid Supports

The present compounds, in certain embodiments, are chemically reactivewherein the compounds comprise a reactive group. In a furtherembodiment, the compounds comprise a carrier molecule or solid support.These substituents, reactive groups, carrier molecules, and solidsupports, comprise a linker that is used to covalently attach thesubstituents to any of the moieties of the present compounds. The solidsupport, carrier molecule or reactive group may be directly attached(where linker is a single bond) to the moieties or attached through aseries of stable bonds, as disclosed above.

Any combination of linkers may be used to attach the carrier molecule,solid support or reactive group and the present compounds together. Thelinker may also be substituted to alter the physical properties of thereporter moiety or chelating moiety, such as spectral properties of thedye. Examples of L include substituted or unsubstituted polyalkylene,arylene, alkylarylene, arylenealkyl, or arylthio moieties.

The linker typically incorporates 1-30 nonhydrogen atoms selected fromthe group consisting of C, N, O, S and P. The linker may be anycombination of stable chemical bonds, optionally including, single,double, triple or aromatic carbon-carbon bonds, as well ascarbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds,sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen bonds,phosphorus-nitrogen bonds, and nitrogen-platinum bonds. Typically thelinker incorporates less than 15 nonhydrogen atoms and are composed ofany combination of ether, thioether, thiourea, amine, ester,carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromaticbonds. Typically the linker is a combination of single carbon-carbonbonds and carboxamide, sulfonamide or thioether bonds. The bonds of thelinker typically result in the following moieties that can be found inthe linker: ether, thioether, carboxamide, thiourea, sulfonamide, urea,urethane, hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl andamine moieties. Examples of a linker include substituted orunsubstituted polymethylene, arylene, alkylarylene, arylenealkyl, andarylthio.

In one embodiment, the linker contains 1-6 carbon atoms; in another, thelinker comprises a thioether linkage. Exemplary linking members includea moiety that includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like.In another embodiment, the linker is or incorporates the formula—(CH₂)_(d)(CONH(CH₂)_(e))_(z)— or where d is an integer from 0-5, e isan integer from 1-5 and z is 0 or 1. In a further embodiment, the linkeris or incorporates the formula —O—(CH₂)—. In yet another embodiment, thelinker is or incorporates a phenylene or a 2-carboxy-substitutedphenylene.

An important feature of the linker is to provide an adequate spacebetween the carrier molecule, reactive group or solid support and thedye so as to prevent steric hinderance. Therefore, the linker of thepresent compound is important for (1) attaching the carrier molecule,reactive group or solid support to the compound, (2) providing anadequate space between the carrier molecule, reactive group or solidsupport and the compound so as not to sterically hinder the action ofthe compound and (3) for altering the physical properties of the presentcompounds.

In another exemplary embodiment of the invention, the present compoundsare chemically reactive, and are substituted by at least one reactivegroup. The reactive group functions as the site of attachment foranother moiety, such as a carrier molecule or a solid support, whereinthe reactive group chemically reacts with an appropriate reactive orfunctional group on the carrier molecule or solid support.

Reactive groups or reactive group precursors may be positioned duringthe formation of the present compounds. Thus, compounds incorporating areactive group can be reacted with and attached to a wide variety ofbiomolecules or non-biomolecules that contain or are modified to containfunctional groups with suitable reactivity. When a labeled componentincludes a compound as disclosed herein, then this conjugate typicallypossesses the nucleic acid staining abilities of the parent compound,particularly RNA staining. However, the present fluorescent compoundscan also function as reporter molecules for the labeled componentswherein the nucleic acid binding properties of the reagents may notemployed.

Preferred reactive groups for incorporation into the disclosed compoundsmay be selected to react with an amine, a thiol or an alcohol. In anexemplary embodiment, the compounds of the invention further comprise areactive group that is an acrylamide, an activated ester of a carboxylicacid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, anazide, an aziridine, a boronate, a diazoalkane, a haloacetamide, ahaloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate,an isothiocyanate, a maleimide, a phosphoramidite, a photoactivatablegroup, a reactive platinum complex, a silyl halide, a sulfonyl halide,and a thiol. In a particular embodiment the reactive group is selectedfrom the group consisting of carboxylic acid, succinimidyl ester of acarboxylic acid, hydrazide, amine and a maleimide. In exemplaryembodiment, at least one member selected from R¹, R², R¹¹, or R¹²comprises a reactive group. Preferably, at least one of R¹ or R¹¹comprises a reactive group or is attached to a reactive group.Alternatively, if the present compound comprises a carrier molecule orsolid support a reactive group may be covalently attached independentlyto those substituents, allowing for further conjugation to a anotherdye, carrier molecule or solid support.

In one aspect, the compound comprises at least one reactive group thatselectively reacts with an amine group. This amine-reactive group isselected from the group consisting of succinimidyl ester, sulfonylhalide, tetrafluorophenyl ester and iosothiocyanates. Thus, in oneaspect, the present compounds form a covalent bond with anamine-containing molecule in a sample. In another aspect, the compoundcomprises at least one reactive group that selectively reacts with athiol group. This thiol-reactive group is selected from the groupconsisting of maleimide, haloalkyl and haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904).

The pro-reactive groups are synthesized during the formation of themonomer moieties and carrier molecule and solid support containingcompounds to provide chemically reactive compounds. In this way,compounds incorporating a reactive group can be covalently attached to awide variety of carrier molecules or solid supports that contain or aremodified to contain functional groups with suitable reactivity,resulting in chemical attachment of the components. In an exemplaryembodiment, the reactive group of the compounds of the invention and thefunctional group of the carrier molecule or solid support compriseelectrophiles and nucleophiles that can generate a covalent linkagebetween them. Alternatively, the reactive group comprises aphotoactivatable group, which becomes chemically reactive only afterillumination with light of an appropriate wavelength. Typically, theconjugation reaction between the reactive group and the carrier moleculeor solid support results in one or more atoms of the reactive groupbeing incorporated into a new linkage attaching the present compound ofthe invention to the carrier molecule or solid support. Selectedexamples of functional groups and linkages are shown in Table 1, wherethe reaction of an electrophilic group and a nucleophilic group yields acovalent linkage.

TABLE 1 Examples of some routes to useful covalent linkagesElectrophilic Group Nucleophilic Group Resulting Covalent Linkageactivated esters* amines/anilines carboxamides acrylamides thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides carboxylic acidsesters alkyl halides thiols thioethers alkyl halides alcohols/phenolsethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides thiols thiophenols aryl halides amines aryl amines aziridinesthiols thioethers boronates glycols boronate esters carbodiimidescarboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acidsesters epoxides thiols thioethers haloacetamides thiols thioethershaloplatinate amino platinum complex haloplatinate heterocycle platinumcomplex haloplatinate thiol platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers halotriazines thiols triazinyl thioethers imido estersamines/anilines amidines isocyanates amines/anilines ureas isocyanatesalcohols/phenols urethanes isothiocyanates amines/anilines thioureasmaleimides thiols thioethers phosphoramidites alcohols phosphite esterssilyl halides alcohols silyl ethers sulfonate esters amines/anilinesalkyl amines sulfonate esters thiols thioethers sulfonate esterscarboxylic acids esters sulfonate esters alcohols ethers sulfonylhalides amines/anilines sulfonamides sulfonyl halides phenols/alcoholssulfonate esters *Activated esters, as understood in the art, generallyhave the formula —COΩ, where Ω is a good leaving group (e.g.succinimidyloxy (—OC₄H₄O₂) sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H),-1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group or aryloxysubstituted one or more times by electron withdrawing substituents suchas nitro, fluoro, chloro, cyano, or trifluoromethyl, or combinationsthereof, used to form activated aryl esters; or a carboxylic acidactivated by a carbodiimide to form an anhydride or mixed anhydride—OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a) and R^(b), which may be thesame or different, are C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆alkoxy; or cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl).**Acyl azides can also rearrange to isocyanates

Choice of the reactive group used to attach the compound of theinvention to the substance to be conjugated typically depends on thereactive or functional group on the substance to be conjugated and thetype or length of covalent linkage desired. The types of functionalgroups typically present on the organic or inorganic substances(biomolecule or non-biomolecule) include, but are not limited to,amines, amides, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, silyl halides, carboxylate esters, sulfonateesters, purines, pyrimidines, carboxylic acids, olefinic bonds, or acombination of these groups. A single type of reactive site may beavailable on the substance (typical for polysaccharides or silica), or avariety of sites may occur (e.g., amines, thiols, alcohols, phenols), asis typical for proteins.

Typically, the reactive group will react with an amine, a thiol, analcohol, an aldehyde, a ketone, or with silica. Preferably, reactivegroups react with an amine or a thiol functional group, or with silica.In one embodiment, the reactive group is an acrylamide, an activatedester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, a silyl halide, an anhydride, an aniline, an arylhalide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine (including hydrazides), animido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, or athiol group. By “reactive platinum complex” is particularly meantchemically reactive platinum complexes such as described in U.S. Pat.No. 5,714,327.

Where the reactive group is an activated ester of a carboxylic acid,such as a succinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester or an isothiocyanates, the resulting compound isparticularly useful for preparing conjugates of carrier molecules suchas proteins, nucleotides, oligonucleotides, or haptens. Where thereactive group is a maleimide, haloalkyl or haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904 (supra)) the resulting compound is particularly useful forconjugation to thiol-containing substances. Where the reactive group isa hydrazide, the resulting compound is particularly useful forconjugation to periodate-oxidized carbohydrates and glycoproteins, andin addition is an aldehyde-fixable polar tracer for cell microinjection.Where the reactive group is a silyl halide, the resulting compound isparticularly useful for conjugation to silica surfaces, particularlywhere the silica surface is incorporated into a fiber optic probesubsequently used for remote ion detection or quantitation.

In a particular aspect, the reactive group is a photoactivatable groupsuch that the group is only converted to a reactive species afterillumination with an appropriate wavelength. An appropriate wavelengthis generally a UV wavelength that is less than 400 nm. This methodprovides for specific attachment to only the target molecules, either insolution or immobilized on a solid or semi-solid matrix.Photoactivatable reactive groups include, without limitation,benzophenones, aryl azides and diazirines.

Preferably, the reactive group is a photoactivatable group, succinimidylester of a carboxylic acid, a haloacetamide, haloalkyl, a hydrazine, anisothiocyanate, a maleimide group, an aliphatic amine, a silyl halide, acadaverine or a psoralen. More preferably, the reactive group is asuccinimidyl ester of a carboxylic acid, a maleimide, an iodoacetamide,or a silyl halide. In a particular embodiment the reactive group is asuccinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester, an iosothiocyanates or a maleimide.

The selection of a covalent linkage to attach the reporter molecule tothe carrier molecule or solid support typically depends on thechemically reactive group on the component to be conjugated. Thediscussion regarding reactive groups in the section immediatelypreceding is relevant here as well. Exemplary reactive groups typicallypresent on the biological or non-biological components include, but arenot limited to, amines, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, sulfonate esters, purines, pyrimidines,carboxylic acids, or a combination of these groups. A single type ofreactive site may be available on the component (typical forpolysaccharides), or a variety of sites may occur (e.g. amines, thiols,alcohols, phenols), as is typical for proteins. A carrier molecule orsolid support may be conjugated to more than one reporter molecule,which may be the same or different, or to a substance that isadditionally modified by a hapten. Although some selectivity can beobtained by careful control of the reaction conditions, selectivity oflabeling is best obtained by selection of an appropriate reactivecompound.

In another exemplary embodiment, the present compound is covalentlybound to a carrier molecule. If the compound has a reactive group, thenthe carrier molecule can alternatively be linked to the compound throughthe reactive group. The reactive group may contain both a reactivefunctional moiety and a linker, or only the reactive functional moiety.

A variety of carrier molecules are useful in the present invention.Exemplary carrier molecules include antigens, steroids, vitamins, drugs,haptens, metabolites, toxins, environmental pollutants, amino acids,peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates,lipids, and polymers. In exemplary embodiment, at least one memberselected from R¹, R², R¹¹, or R¹² comprises a carrier molecule.Preferably, at least one of R¹ or R¹¹ comprises a carrier molecule or isattached to a carrier molecule. Alternatively, if the present compoundcomprises a reactive group or solid support a reactive group may becovalently attached independently to those substituents, allowing forfurther conjugation to a reactive group, carrier molecule or solidsupport.

In an exemplary embodiment, the carrier molecule comprises an aminoacid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof. In another exemplary embodiment, the carrier molecule isselected from a hapten, a nucleotide, an oligonucleotide, a nucleic acidpolymer, a protein, a peptide or a polysaccharide. In a preferredembodiment the carrier molecule is amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a tyramine, a synthetic polymer, a polymeric microparticle, abiological cell, cellular components, an ion chelating moiety, anenzymatic substrate or a virus. In another preferred embodiment, thecarrier molecule is an antibody or fragment thereof, an antigen, anavidin or streptavidin, a biotin, a dextran, an antibody bindingprotein, a fluorescent protein, agarose, and a non-biologicalmicroparticle. Typically, the carrier molecule is an antibody, anantibody fragment, antibody-binding proteins, avidin, streptavidin, atoxin, a lectin, or a growth factor. Preferred haptens include biotin,digoxigenin and fluorophores.

Antibody binging proteins include, but are not limited to, protein A,protein G, soluble Fc receptor, protein L, lectins, anti-IgG, anti-IgA,anti-IgM, anti-IgD, anti-IgE or a fragment thereof.

In an exemplary embodiment, the enzymatic substrate is selected from anamino acid, peptide, sugar, alcohol, alkanoic acid, 4-guanidinobenzoicacid, nucleic acid, lipid, sulfate, phosphate, —CH₂OCOalkyl andcombinations thereof. Thus, the enzyme substrates can be cleave byenzymes selected from the group consisting of peptidase, phosphatase,glycosidase, dealkylase, esterase, guanidinobenzotase, sulfatase,lipase, peroxidase, histone deacetylase, endoglycoceramidase,exonuclease, reductase and endonuclease.

In another exemplary embodiment, the carrier molecule is an amino acid(including those that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In a related embodiment, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Exemplary peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other exemplary peptides may function as organellelocalization peptides, that is, peptides that serve to target theconjugated compound for localization within a particular cellularsubstructure by cellular transport mechanisms. Preferred protein carriermolecules include enzymes, antibodies, lectins, glycoproteins, histones,albumins, lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins andgrowth factors. Typically, the protein carrier molecule is an antibody,an antibody fragment, avidin, streptavidin, a toxin, a lectin, or agrowth factor. Exemplary haptens include biotin, digoxigenin andfluorophores.

In another exemplary embodiment, the carrier molecule comprises anucleic acid base, nucleoside, nucleotide or a nucleic acid polymer,optionally containing an additional linker or spacer for attachment of afluorophore or other ligand, such as an alkynyl linkage (U.S. Pat. No.5,047,519), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or otherlinkage. In another exemplary embodiment, the nucleotide carriermolecule is a nucleoside or a deoxynucleoside or a dideoxynucleoside.

Exemplary nucleic acid polymer carrier molecules are single- ormulti-stranded, natural or synthetic DNA or RNA oligonucleotides, orDNA/RNA hybrids, or incorporating an unusual linker such as morpholinederivatized phosphates (AntiVirals, Inc., Corvallis Oreg.), or peptidenucleic acids such as N-(2-aminoethyl)glycine units, where the nucleicacid contains fewer than 50 nucleotides, more typically fewer than 25nucleotides.

In another exemplary embodiment, the carrier molecule comprises acarbohydrate or polyol that is typically a polysaccharide, such asdextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch,agarose and cellulose, or is a polymer such as a poly(ethylene glycol).In a related embodiment, the polysaccharide carrier molecule includesdextran, agarose or FICOLL.

In another exemplary embodiment, the carrier molecule comprises a lipid(typically having 6-25 carbons), including glycolipids, phospholipids,and sphingolipids. Alternatively, the carrier molecule comprises a lipidvesicle, such as a liposome, or is a lipoprotein (see below). Somelipophilic substituents are useful for facilitating transport of theconjugated dye into cells or cellular organelles.

Alternatively, the carrier molecule is cells, cellular systems, cellularfragments, or subcellular particles. Examples of this type of conjugatedmaterial include virus particles, bacterial particles, virus components,biological cells (such as animal cells, plant cells, bacteria, oryeast), or cellular components. Examples of cellular components that canbe labeled, or whose constituent molecules can be labeled, include butare not limited to lysosomes, endosomes, cytoplasm, nuclei, histones,mitochondria, Golgi apparatus, endoplasmic reticulum and vacuoles.

In another embodiment the carrier molecule is a metal chelating moiety.While any chelator that binds a metal ion of interest and gives a changein its fluorescence properties is a suitable conjugate, preferred metalchelating moieties are crown ethers, including diaryldiaza crown ethers,as described in U.S. Pat. No. 5,405,975 to Kuhn et al. (1995);derivatives of 1,2-bis-(2-aminophenoxyethane)-N,N,N′,N′-tetraacetic acid(BAPTA), as described in U.S. Pat. No. 5,453,517 to Kuhn et al. (1995)(incorporated by reference) and U.S. Pat. No. 5,049,673 to Tsien et al.(1991); derivatives of 2-carboxymethoxy-aniline-N,N-diacetic acid(APTRA), as described by Ragu et al., Am. J. Physiol., 256: C540 (1989);and pyridyl-based and phenanthroline metal ion chelators, as describedin U.S. Pat. No. 5,648,270 to Kuhn et al. (1997).

Fluorescent conjugates of metal chelating moieties possess utility asindicators for the presence of a desired metal ion. While fluorescention-indicators are known in the art, the incorporation of thefluorinated fluorogenic and fluorescent compounds of the presentinvention imparts the highly advantageous properties of the instantfluorophores onto the resulting ion indicator.

The ion-sensing conjugates of the invention are optionally prepared inchemically reactive forms and further conjugated to polymers such asdextrans to improve their utility as sensors as described in U.S. Pat.Nos. 5,405,975 and 5,453,517.

In another exemplary embodiment, the carrier molecule non-covalentlyassociates with organic or inorganic materials. Exemplary embodiments ofthe carrier molecule that possess a lipophilic substituent can be usedto target lipid assemblies such as biological membranes or liposomes bynon-covalent incorporation of the dye compound within the membrane,e.g., for use as probes for membrane structure or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials.

In an exemplary embodiment, the carrier molecule comprises a specificbinding pair member wherein the present compounds are conjugated to aspecific binding pair member and are used to detect an analyte in asample. Alternatively, the presence of the labeled specific binding pairmember indicates the location of the complementary member of thatspecific binding pair; each specific binding pair member having an areaon the surface or in a cavity which specifically binds to, and iscomplementary with, a particular spatial and polar organization of theother. Exemplary binding pairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) aDNA (aRNA)†hormone hormone receptor ion chelator antibody antibody-binding proteins*IgG is an immunoglobulin †cDNA and cRNA are the complementary strandsused for hybridization

In an exemplary embodiment, the present compounds of the invention arecovalently bonded to a solid support. The solid support may be attachedto the compound or through a reactive group, if present, or through acarrier molecule, if present. In exemplary embodiment, at least onemember selected from R¹, R², R¹¹, or R¹² comprises a solid support.Preferably, at least one of R¹ or R¹¹ comprises a solid support or isattached to a solid support. Alternatively, if the present compoundcomprises a carrier molecule or reactive group a solid support may becovalently attached independently to those substituents, allowing forfurther conjugation to a another dye, carrier molecule or solid support.

A solid support suitable for use in the present invention is typicallysubstantially insoluble in liquid phases. Solid supports of the currentinvention are not limited to a specific type of support. Rather, a largenumber of supports are available and are known to one of ordinary skillin the art. Thus, useful solid supports include solid and semi-solidmatrixes, such as aerogels and hydrogels, resins, beads, biochips(including thin film coated biochips), microfluidic chip, a siliconchip, multi-well plates (also referred to as microtitre plates ormicroplates), membranes, conducting and nonconducting metals, glass(including microscope slides) and magnetic supports. More specificexamples of useful solid supports include silica gels, polymericmembranes, particles, derivatized plastic films, glass beads, cotton,plastic beads, alumina gels, polysaccharides such as Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride,polypropylene, polyethylene (including poly(ethylene glycol)), nylon,latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead,starch and the like.

In some embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, etc., for attaching the compounds of the invention. Usefulreactive groups are disclosed above and are equally applicable to thesolid support reactive functional groups herein.

A suitable solid phase support can be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the compounds of theinvention to the solid support, resins generally useful in peptidesynthesis may be employed, such as polystyrene (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

Preparation of Conjugates

Conjugates of components (carrier molecules and solid supports), e.g.,drugs, peptides, toxins, nucleotides, phospholipids and other organicmolecules are prepared by organic synthesis methods using the reactivedyes, are generally prepared by means well recognized in the art(Haugland, MOLECULAR PROBES HANDBOOK, supra, 2002).

Conjugation to form a covalent bond may consist of simply mixing thereactive dyes of the present invention in a suitable solvent in whichboth the reactive compound and the substance to be conjugated aresoluble. The reaction preferably proceeds spontaneously without addedreagents at room temperature or below. For those reactive dyes that arephotoactivated, conjugation is facilitated by illumination of thereaction mixture to activate the reactive dye. Chemical modification ofwater-insoluble substances, so that a desired dye-conjugate may beprepared, is preferably performed in an aprotic solvent such asdimethylformamide (DMF), dimethylsulfoxide (DMSO), acetone, ethylacetate, toluene, or chloroform.

Preparation of peptide or protein conjugates typically comprises firstdissolving the protein to be conjugated in aqueous buffer at about. 1-10mg/mL at room temperature or below. Bicarbonate buffers (pH about 8.3)are especially suitable for reaction with succinimidyl esters, phosphatebuffers (pH about 7.2-8) for reaction with thiol-reactive functionalgroups and carbonate or borate buffers (pH about 9) for reaction withisothiocyanates and dichlorotriazines. The appropriate reactive dye isthen dissolved in a nonhydroxylic solvent (usually DMSO or DMF) in anamount sufficient to give a suitable degree of conjugation when added toa solution of the protein to be conjugated. The appropriate amount ofcompound for any protein or other component is convenientlypredetermined by experimentation in which variable amounts of the dyeare added to the protein, the conjugate is chromatographically purifiedto separate unconjugated compound and the compound-protein conjugate istested in its desired application.

Following addition of the reactive compound to the component solution,the mixture may be incubated for a suitable period (typically about 1hour at room temperature to several hours on ice), the excess unreactedcompound is removed by gel filtration, dialysis, HPLC, adsorption on anion exchange or hydrophobic polymer or other suitable means. Theconjugate is used in solution or lyophilized. In this way, suitableconjugates can be prepared from antibodies, antibody fragments, avidins,lectins, enzymes, proteins A and G, cellular proteins, albumins,histones, growth factors, hormones, and other proteins. The approximatedegree of substitution is determined from the long wavelength absorptionof the compound-protein conjugate by using the extinction coefficient ofthe un-reacted compound at its long wavelength absorption peak, theunmodified protein's absorption peak in the ultraviolet and bycorrecting the UV absorption of the conjugate for absorption by thecompound in the UV.

Conjugates of polymers, including biopolymers and other higher molecularweight polymers are typically prepared by means well recognized in theart (for example, Brinkley et al., Bioconjugate Chem., 3: 2 (1992)). Inthese embodiments, a single type of reactive site may be available, asis typical for polysaccharides or multiple types of reactive sites (e.g.amines, thiols, alcohols, phenols) may be available, as is typical forproteins. Selectivity of labeling is best obtained by selection of anappropriate reactive dye. For example, modification of thiols with athiol-selective reagent such as a haloacetamide or maleimide, ormodification of amines with an amine-reactive reagent such as anactivated ester, acyl azide, isothiocyanate or3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained bycareful control of the reaction conditions.

When modifying polymers with the compounds, an excess of the compound istypically used, relative to the expected degree of dye substitution. Anyresidual, un-reacted compound or hydrolysis product is typically removedby dialysis, chromatography or precipitation. Presence of residual,unconjugated compound can be detected by thin layer chromatography usinga solvent that elutes the compound away from its conjugate. In all casesit is usually preferred that the reagents be kept as concentrated aspractical so as to obtain adequate rates of conjugation.

In an exemplary embodiment, the conjugate is associated with anadditional substance that binds either to the compound or the labeledcomponent through noncovalent interaction. In another exemplaryembodiment, the additional substance is an antibody, an enzyme, ahapten, a lectin, a receptor, an oligonucleotide, a nucleic acid, aliposome, or a polymer. The additional substance is optionally used toprobe for the location of the conjugate, for example, as a means ofenhancing the signal of the conjugate.

Methods of Use

The present nucleic acid reporter molecules may be utilized withoutlimit for the fluorescent detection of nucleic acid polymers in a testsample. The methods for the detection of single, double, triple orquadruple stranded DNA and RNA or a combination thereof comprisescontacting a sample with a present nucleic acid reporter molecule toprepare a labeling mixture, incubating the sample with the stainingsolution for a sufficient amount of time for the present reportermolecules to complex with the nucleic acid, illuminating the sample withan appropriate wavelength and observing the illuminated labeling mixturewhereby the nucleic acid polymer is detected.

The compound is typically combined with the sample as a stainingsolution. The staining solution is typically prepared by dissolving apresent nucleic acid reporter molecule in an aqueous solvent such aswater, a buffer solution or assay solution, such as phosphate bufferedsaline, or an organic solvent such as dimethylsulfoxide (DMSO),dimethylformamide (DMF), methanol, ethanol or acetonitrile. Typically,the present nucleic reporter molecules are first dissolved in an organicsolvent such as DMSO as a stock solution. The stock solution istypically prepared about 100-300× more concentrated that the effectiveworking concentration. Thus, the stock solution is diluted to aneffective working concentration in an aqueous solution that optionallyincludes appropriate buffering components and a detergent. An effectiveworking concentration of the present compounds is the amount sufficientto give a detectable optical response when complexed with nucleic acidpolymers. Typically, the effective amount is about 10 nM to 10 μM. Mostpreferred is about 20 nM to 1 μM. For selected reporter compounds,staining is optimal when the staining solution has a concentration ofabout 0.04 μM to about 0.1 μM. It is generally understood that thespecific amount of the nucleic acid reporter molecules present in astaining solution is determined by the physical nature of the sample andthe nature of the analysis being performed.

In an exemplary embodiment, the staining solution contains a detergent.This is particularly useful when the nucleic acid is present in anaqueous sample solution. Without wishing to be bound by a theory itappears that a low concentration of detergent stabilizes the presentnucleic acid reporter molecule when present in solution. Thus thestaining solution can be combined with an aqueous sample providing anoptimized solution based detection assay. Detergents include, but arenot limited to, CHAPS, Triton-X, SDS and Tween 20. The detergent istypically present in an aqueous solution at a concentration from about0.0001% to about 0.2% (w/v). More specifically the detergent is presentfrom about 0.0015% to about 0.005% (w/v). In an exemplary embodiment astaining solution comprises a present nucleic acid reporter moleculepresent at about 0.04 μM and the detergent CHAPS present at about0.0025% (w/v).

In a further embodiment, the present staining solution contains an agentto reduce the signal to noise ratio. Such an agent improves thelinearity of the curve obtained from a range of nucleic acidconcentrations. Thus, in an exemplary embodiment, a staining solutionfor detecting RNA in the presence of DNA, contains a low concentrationof DNA. Without wishing to be bound by a theory it appears that this lowconcentration associates with the present reporter molecules, which donot fluoresce, and lowers the background fluorescent signal. Preferablythis DNA is double stranded and in one aspect the DNA is lambda DNA.Thus, in one embodiment, the present staining solution contains DNA in aconcentration from about 1.0 μg/ml to about 0.05 μg/ml. Morespecifically the DNA is present about 0.05 μg/ml to about 0.01 μg/ml.Therefore, in an exemplary embodiment a present staining solutioncomprises a present nucleic acid reporter molecule present at about 0.04μM and DNA at a concentration of about 0.2 μg/ml. In a furtherembodiment this staining solution would also include a detergent, asdisclosed above.

Therefore, the present disclosure provides a staining solution thatcomprises a present nucleic acid reporter molecule in a buffer solution.In a further embodiment this staining solution comprises a detergent andin yet a further embodiment, the staining solution also comprises a lowconcentration of DNA.

The sample may be combined with the staining solution by any means thatfacilitates contact between the nucleic acid reporter molecules and thenucleic acid. The contact can occur through simple mixing, as in thecase where the sample is a solution. The present reporter molecules maybe added to the nucleic acid solution directly or may contact thesolution on an inert matrix such as a blot or gel, a testing strip, amicroarray, or any other solid or semi-solid surface, for example whereonly a simple and visible demonstration of the presence of nucleic acidsis desired. Any inert matrix used to separate the sample can be used todetect the presence of nucleic acids by observing the fluorescentresponse on the inert matrix.

Thus, in one embodiment is provided a composition comprising a sampleand a present nucleic acid reporter molecule. In one aspect thiscomposition is an aqueous solution.

Alternatively, the sample may include cells and/or cell membranes. Whileselected examples of the compound disclosed herein may permeate cellularmembranes rapidly and completely upon addition of the staining solution,any technique that is suitable for transporting the reporter moleculesacross cell membranes with minimal disruption of the viability of thecell and integrity of cell membranes is a valid method of combining thesample with the present reporter molecules for detection ofintracellular nucleic acid. Examples of suitable processes includeaction of chemical agents such as detergents, enzymes or adenosinetriphosphate; receptor- or transport protein-mediated uptake;pore-forming proteins; microinjection; electroporation; hypoosmoticshock; or minimal physical disruption such as scrape loading orbombardment with solid particles coated with or in the presence of thepresent reporter molecules.

The sample is incubated in the presence of the nucleic acid reportermolecules for a time sufficient to form the fluorescent nucleicacid-reporter molecule complex. Detectable fluorescence in a solution ofnucleic acids is essentially instantaneous. Detectable fluorescencewithin cell membranes requires the permeation of the dye into the cell.While most present nucleic acid reporter molecules are not cell permeantdue to the presence of at least one negatively charged moiety, it isenvisioned that the present compounds could be adequately substituted toprovide cell permeant versions of the present compounds. In general,visibly detectable fluorescence can be obtained in a wide variety ofcells with certain cell permeant embodiments of the present inventionwithin about 10-30 minutes after combination with the sample, commonlywithin about 10-20 minutes. While permeation and fluorescence should berapid for all reporter molecules comprising an aromatic substituent onthe pyridinium or quinolinium moiety of the D moiety, it is readilyapparent to one skilled in the art that the time necessary forsufficient permeation of the dye, or sufficient formation of thefluorescent nucleic acid complex, is dependent upon the physical andchemical nature of the individual sample and the sample medium.

In another embodiment, is provided a complex comprising a presentnucleic acid reporter molecule and a nucleic acid polymer. To facilitatethe detection of the nucleic acid-reporter molecule complex, theexcitation or emission properties of the fluorescent complex areutilized. For example, the sample is excited by a light source capableof producing light at or near the wavelength of maximum absorption ofthe fluorescent complex, such as an ultraviolet or visible lamp, an arclamp, a laser, or even sunlight. Preferably the fluorescent complex isexcited at a wavelength equal to or greater than about 300 nm, morepreferably equal to or greater than about 340 nm. The fluorescence ofthe complex is detected qualitatively or quantitatively by detection ofthe resultant light emission at a wavelength of greater than about 400nm, more preferably greater than about 450 nm, most preferred greaterthan 480 nm. The emission is detected by means that include visibleinspection, photographic film, or the use of current instrumentationsuch as fluorometers, quantum counters, plate readers, epifluorescencemicroscopes and flow cytometers or by means for amplifying the signalsuch as a photomultiplier.

In an exemplified embodiment, the present nucleic acid reportercompounds are used to detect RNA in the presence of DNA, wherein themethod comprises the following steps:

-   a. combining a present nucleic acid reporter molecule with a sample    to prepare a labeling mixture, wherein the nucleic acid reporter    molecule has a RNA/DNA ratio of fluorescence enhancement greater    than about one;-   b. incubating the labeling mixture for a sufficient amount of time    for the nucleic acid reporter molecule to associate with RNA in the    sample to form an incubated mixture;-   c. illuminating the incubated mixture with an appropriate wavelength    to form an illuminated mixture; and,-   d. observing the illuminated mixture whereby the RNA is detected in    the presence of DNA.

Typically, the fluorescence of the RNA complex is distinguishable fromthe fluorescence of a DNA complex with the compound. This difference maybe due to any detectable optical property, but in one embodiment, thefluorescence of the RNA complex is brighter than the fluorescence of acorresponding DNA complex with the compound. Therefore, in an exemplaryembodiment, by comparing the fluorescence response of the RNA complexwith a standard, the amount of RNA in the sample may be quantitated,even in the presence of DNA.

As discussed above, the RNA present in the sample may be present in asolution, or in or on a solid or semisolid support. In a preferredembodiment, the nucleic acid is present in an aqueous solution. Thedetection of RNA in solution may also be enhanced by the addition of adetergent to the staining solution. Exemplified detergents include, butare not limited to CHAPS, Triton-X, SDS and Tween 20. Particularlypreferred is CHAPS, wherein the fluorescent single in an aqueous signalis stabilized for at least 6 hours. The detection of RNA in a solutionmay also be further enhanced by the addition of a low concentration ofDNA to the staining solution.

Typically, the fluorescence of the RNA complex is distinguishable fromthe fluorescence of a DNA complex with the compound by fluorescenceintensity. This difference may be due to any detectable opticalproperty, but in one embodiment, the fluorescence of the RNA complex isbrighter than the fluorescence of a corresponding DNA complex with thecompound.

By comparing the fluorescence response of the RNA complex with astandard, the amount of RNA in the sample may be quantitated, even inthe presence of DNA.

As discussed above, the RNA present in the sample may be present in asolution, or in or on a solid or semisolid support. The RNA itself maybe selected from one or more of mRNA, tRNA, and rRNA.

The method may also be enhanced by the addition of an additionaldetection reagent that exhibits a greater fluorescence response whenassociated with DNA than when associated with RNA. A variety of nucleicacid stains that fluoresce brightly when complexed with DNA are known inthe art.

The present nucleic acid reporter molecules that are capable ofproducing a fluorescent intensity signal that is greater on RNA than onDNA are determined empirically. The relative selectivity of the presentcompounds for differentiating RNA and DNA may be readily evaluated asset out in Examples 2, and 6-12.

The foregoing methods having been described it is understood that themany and varied compounds of the present invention can be utilized withthe many methods. The compounds not being limited to just those that arespecifically disclosed.

In an exemplary embodiment the present methods employ a nucleic acidreporter molecule that comprises the formula

wherein at least one of R³, R⁴, and R⁵ is an alkyl, substituted alkyl, a5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, asubstituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or7-membered aryl or a substituted 5-, 6- or 7-membered aryl; and theremaining R³, R⁴ or R⁵ are hydrogen.

In an exemplary embodiment at least one of R³, R⁴, and R⁵ is asubstituted alkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl,a substituted 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or7-membered heteroaryl, or a substituted 5-, 6- or 7-membered aryl thatis substituted by an alkyl, —(CH₂)_(k)—NR⁶R⁷; —COOR⁸, NO₂, or halogen,wherein k is an integer from 0 to about 6. The substituents R⁶, R⁷ andR⁸ are independently hydrogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, sulfoalkyl or aminoalkyl. In one aspect at least oneof R³ and R⁴ is a thiophenyl, substituted thiophenyl, adamantyl,substituted adamantly, phenyl, substituted phenyl, alkyl, substitutedalkyl, benzyl or substituted benzyl.

These nucleic acid reporter molecules exhibit a fluorescence enhancementwhen non-covalently associated with a nucleic acid molecule. In oneaspect, the fluorescence enhancement is greater when the nucleic acid isRNA than when the nucleic acid is DNA. In another aspect, thefluorescence enhancement is greater when the nucleic acid is DNA thanwhen the nucleic acid is RNA.

The R¹ substituent is independently hydrogen, carboxy, sulfo, phosphate,phosphonate, amino, hydroxyl, trifluoromethyl, halogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, alkylamino, substitutedalkylamino, dialkylamino, substituted dialkylamino, aminoalkyl,substituted aminoalkyl, fused benzene, substituted fused benzene,reactive group, solid support or carrier molecule and t is an integerfrom 1 to 4. In one aspect R¹ is alkoxy or halogen. In another aspect R¹is methoxy, Br, Cl, or F.

The R² substituent is an alkyl, substituted alkyl, arylalkyl,substituted arylalkyl, heteroalkyl, substituted heteroalkyl, alkoxy,substituted alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo,sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, ortrialkylammonium. In one aspect R² is methyl, ethyl, propyl, or—(CH₂)₃SO₃ ⁻.

X is O, S or Se.

The D is a substituted pyridinium, unsubstituted pyridinium, substitutedquinolinium, unsubstituted quinolinium, substituted benzazolium orunsubstituted benzazolium moiety. In an exemplary embodiment, D has theformula:

wherein R¹¹ is hydrogen, substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl,unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl,substituted heteroarylalkyl; unsubstituted heteroarylalkyl, substitutedheteroaryl, unsubstituted heteroaryl substituted cycloalkyl,unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstitutedheterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstitutedalkylamino, substituted alkylthio, unsubstituted alkylthio, reactivegroup, solid support, or carrier molecule and R¹² is an alkyl,substituted alkyl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, alkoxy, substituted alkoxy, carboxy,carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino,aminoalkyl, alkylamino, dialkylamino, or trialkylammonium.Alternatively, R¹¹ in combination with an adjacent R¹¹ or R¹², togetherwith the atoms to which they are joined, form a ring which is a 5-, 6-or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-memberedheterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, a substituted 5-,6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heteroaryl, asubstituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or 7-membered arylor a substituted 5-, 6- or 7-membered aryl.

In an exemplary embodiment of the methods, the nucleic acid reportermolecule has the formula:

-   -   or the formula

In an exemplary embodiment, the nucleic aid reporter molecule of thepresent methods comprise a reactive group, solid support and carriermolecule wherein these substituents independently comprise a linker thatis a single covalent bond, or a covalent linkage that is linear orbranched, cyclic or heterocyclic, saturated or unsaturated, having 1-20nonhydrogen atoms selected from the group consisting of C, N, P, O andS; and are composed of any combination of ether, thioether, amine,ester, carboxamide, sulfonamide, hydrazide bonds and aromatic orheteroaromatic bonds.

In an exemplary embodiment, the reactive group is an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a silyl halide, a sulfonylhalide, a thiol or a photoactivatable group. In a further aspect, thereactive group is carboxylic acid, succinimidyl ester of a carboxylicacid, hydrazide, amine or a maleimide.

In an exemplary embodiment the carrier molecule is an amino acid, apeptide, a protein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid polymer, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell or a virus. In a further aspect, thecarrier molecule is an antibody or fragment thereof, an avidin orstreptavidin, a biotin, a blood component protein, a dextran, an enzyme,an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescentprotein, a growth factor, a lectin, a lipopolysaccharide, amicroorganism, a metal binding protein, a metal chelating moiety, anon-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, or a tyramide.

In an exemplary embodiment, the solid support is a microfluidic chip, asilicon chip, a microscope slide, a microplate well, silica gels,polymeric membranes, particles, derivatized plastic films, glass beads,cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride,polypropylene, polyethylene, nylon, latex bead, magnetic bead,paramagnetic bead, or superparamagnetic bead. In a further aspect, thesolid support is Sepharose, poly(acrylate), polystyrene,poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch,FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose,diazocellulose or starch.

Sample Preparation

The sample may be prepared using methods well known in the art forisolating nucleic acid for in vitro and solution based assay detectionor well know methods for live cell or fixed cells for intracellularand/or in vivo detection of nucleic acids. The sample includes, withoutlimitation, any biological derived material that is thought to contain anucleic acid polymer. Alternatively, samples also include material thatnucleic acid polymers have been added to such as a PCR reaction mixture,a polymer gel such as agarose or polyacrylamide gels or a microfluidicassay system. In another aspect of the present disclosure, the samplecan also include a buffer solution that contains nucleic acid polymersto determine the present reporter molecules that are ideal underdifferent assay conditions or to determine the present reportermolecules that are preferential RNA reporters.

The sample can be a biological fluid such as whole blood, plasma, serum,nasal secretions, sputum, saliva, urine, sweat, transdermal exudates,cerebrospinal fluid, or the like. Biological fluids also include tissueand cell culture medium wherein an analyte of interest has been secretedinto the medium. Alternatively, the sample may be whole organs, tissueor cells from the animal. Examples of sources of such samples includemuscle, eye, skin, gonads, lymph nodes, heart, brain, lung, liver,kidney, spleen, thymus, pancreas, solid tumors, macrophages, mammaryglands, mesothelium, and the like. Cells include without limitationprokaryotic cells such as bacteria, yeast, fungi, mycobacteria andmycoplasma, and eukaryotic cells such as nucleated plant and animalcells that include primary cultures and immortalized cell lines.Typically prokaryotic cells include E. coli and S. aureus. Eukaryoticcells include without limitation ovary cells, epithelial cells,circulating immune cells, p cells, hepatocytes, and neurons.

In an exemplary embodiment, the sample comprises biological fluids,buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryoticcells, nucleic acid polymers, nucleosides, nucleotides, a polymeric gelor tissue sections. In a further aspect, the sample comprises nucleicacid polymers in an aqueous buffer.

The nucleic acid may be either natural (biological in origin) orsynthetic (prepared artificially). The nucleic acid may be present asnucleic acid fragments, oligonucleotides, or nucleic acid polymers. Thenucleic acid may be present in a condensed phase, such as a chromosome.The presence of the nucleic acid in the sample may be due to asuccessful or unsuccessful experimental methodology, undesirablecontamination, or a disease state. Nucleic acid may be present in all,or only part, of a sample, and the presence of nucleic acids may be usedto distinguish between individual samples, or to differentiate a portionor region within a single sample.

The nucleic acid may be enclosed in a biological structure, for examplecontained within a viral particle, an organelle, or within a cell. Thenucleic acids enclosed in biological structures may be obtained from awide variety of environments, including cultured cells, organisms ortissues, unfiltered or separated biological fluids such as urine,cerebrospinal fluid, blood, lymph fluids, tissue homogenate, mucous,saliva, stool, or physiological secretions or environmental samples suchas soil, water and air. The nucleic acid may be endogenous or introducedas foreign material, such as by infection or by transfection. Thepresent nucleic acid reporter molecules can also be used for stainingnucleic acids in a cell or cells that is fixed and treated with routinehistochemical or cytochemical procedures.

Alternatively, the nucleic acid is not enclosed within a biologicalstructure, but is present as a sample solution. The sample solution canvary from one of purified nucleic acids to crude mixtures such as cellextracts, biological fluids and environmental samples. In some cases itis desirable to separate the nucleic acids from a mixture ofbiomolecules or fluids in the solution prior to combination with thepresent reporter molecules. Numerous, well known, techniques exist forseparation and purification of nucleic acids from generally crudemixtures with other proteins or other biological molecules. Theseinclude such means as electrophoretic techniques and chromatographictechniques using a variety of supports.

The sample may be incubated in the presence of the nucleic acid reportermolecules for a time sufficient to form a nucleic acid-reporter moleculecomplex. While permeation and complexation may be more or less rapid forthe compounds disclosed herein, largely depending on the nature of thesubstituents present on the compound. It should be apparent to oneskilled in the art that the time necessary for sufficient permeation ofthe dye, or sufficient formation of the resulting nucleic acid complex,is dependent upon the physical and chemical nature of the individualsample and the sample medium (see for example U.S. Pat. No. 5,658,751).

Illumination

The sample containing a nucleic acid-reporter molecule complex may beilluminated with a wavelength of light selected to give a detectableoptical response, and observed with a means for detecting the opticalresponse. By optical response is meant any detectable colorimetric orluminescent property of the complex. Typically, the optical response isrelated to the excitation or emission properties of the complex.

For example, the sample may be excited by a light source capable ofproducing light at or near the wavelength of maximum absorption of thefluorescent complex, such as an ultraviolet or visible lamp, an arclamp, a laser, or even sunlight. The optical response is optionallydetected by visual inspection, or by use of any of the followingdevices: CCD camera, video camera, photographic film, laser-scanningdevices, fluorometers, photodiodes, quantum counters, epifluorescencemicroscopes, scanning microscopes, flow cytometers, fluorescencemicroplate readers, or by means for amplifying the signal such asphotomultiplier tubes. Where the sample is examined using a flowcytometer, examination of the sample optionally includes sortingportions of the sample according to their fluorescence response.

The wavelengths of the excitation and emission bands of the nucleic acidreporter molecules vary with reporter molecule composition to encompassa wide range of illumination and detection bands. This allows theselection of individual reporter molecules for use with a specificexcitation source or detection filter. In particular, present reportermolecules can be selected that possess excellent correspondence of theirexcitation band with the 633/647 nm band of the commonly used argon-ionlaser or emission bands which are coincident with preexisting filters.

The presence, location, and distribution of nucleic acid, particularlyRNA, may be detected using the spectral properties of thecompound-nucleic acid complex. Once the dye-nucleic acid complex isformed, its presence may be detected and used as an indicator of thepresence, location, or type of nucleic acids in the sample, or as abasis for sorting cells, or as a key to characterizing the sample orcells in the sample. Such characterization may be enhanced by the use ofadditional reagents, including fluorescent reagents. The nucleic acidconcentration in a sample can also be quantified by comparison withknown relationships between the fluorescence of the nucleic acid-dyecomplex and concentration of nucleic acids in the sample. In particular,fluorescence intensity may be compared to a standard curve prepared fromsamples containing known nucleic acid concentrations, particularly RNAconcentrations.

Kits

Suitable kits for forming a nucleic acid-reporter molecule complex anddetecting the nucleic acid also form part of the present disclosure.Such kits can be prepared from readily available materials and reagentsand can come in a variety of embodiments. The contents of the kit willdepend on the design of the assay protocol or reagent for detection ormeasurement. All kits will contain instructions, appropriate reagents,and one or more of the presently disclosed nucleic acid reportermolecules. Typically, instructions include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to be addedtogether, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like to allow the user to carryout any one of the methods or preparations described above. In oneaspect, the kit is formulated to facilitate the high-throughputscreening of multiple samples, such as may be accomplished usingautomated methods.

Thus, a kit for detecting nucleic acid in a sample may comprise acompound as described above. The kit may further include instructionsfor performing one or more of the above disclosed methods, including thedetection and/or quantitation of RNA in the presence of DNA.

The kit may optionally further include one or more of the following;sample preparation reagents, a buffering agent, a nucleic acid reportermolecule dilution buffer, an organic solvent or an additional detectionreagent, particularly where the additional detection reagent is anadditional distinct nucleic acid reporter molecule. Where the additionalnucleic acid reporter is a DNA-selective nucleic acid stain, the kit maybe useful for detecting and/or quantitating RNA in the presence of DNA.In one aspect the dilution buffer (for the present reporter molecules)comprises a detergent and/or a low concentration of DNA.

In an exemplary embodiment the present kits comprise a nucleic acidreporter molecule that comprises the formula

wherein at least one of R³, R⁴, and R⁵ is an alkyl, substituted alkyl, a5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, asubstituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or7-membered aryl or a substituted 5-, 6- or 7-membered aryl; and theremaining R³, R⁴ or R⁵ are hydrogen.

In an exemplary embodiment at least one of R³, R⁴, and R⁵ is asubstituted alkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl,a substituted 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or7-membered heteroaryl, or a substituted 5-, 6- or 7-membered aryl thatis substituted by an alkyl, —(CH₂)_(k)—NR⁶R⁷; —COOR⁸, NO₂, or halogen,wherein k is an integer from 0 to about 6. The substituents R⁶, R⁷ andR⁸ are independently hydrogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, sulfoalkyl or aminoalkyl. In one aspect at least oneof R³ and R⁴ is a thiophenyl, substituted thiophenyl, adamantyl,substituted adamantly, phenyl, substituted phenyl, alkyl, substitutedalkyl, benzyl or substituted benzyl.

These nucleic acid reporter molecules exhibit a fluorescence enhancementwhen non-covalently associated with a nucleic acid molecule. In oneaspect, the fluorescence enhancement is greater when the nucleic acid isRNA than when the nucleic acid is DNA. In another aspect, thefluorescence enhancement is greater when the nucleic acid is DNA thanwhen the nucleic acid is RNA.

The R¹ substituent is independently hydrogen, carboxy, sulfo, phosphate,phosphonate, amino, hydroxyl, trifluoromethyl, halogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, alkylamino, substitutedalkylamino, dialkylamino, substituted dialkylamino, aminoalkyl,substituted aminoalkyl, fused benzene, substituted fused benzene,reactive group, solid support or carrier molecule and t is an integerfrom 1 to 4. In one aspect R¹ is alkoxy or halogen. In another aspect R¹is methoxy, Br, Cl, or F.

The R² substituent is an alkyl, substituted alkyl, arylalkyl,substituted arylalkyl, heteroalkyl, substituted heteroalkyl, alkoxy,substituted alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo,sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, ortrialkylammonium. In one aspect R² is methyl, ethyl, propyl, or—(CH₂)₃SO₃ ⁻.

X is O, S or Se.

The D is a substituted pyridinium, unsubstituted pyridinium, substitutedquinolinium, unsubstituted quinolinium, substituted benzazolium orunsubstituted benzazolium moiety. In an exemplary embodiment, D has theformula:

wherein R¹¹ is hydrogen, substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl,unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl,substituted heteroarylalkyl; unsubstituted heteroarylalkyl, substitutedheteroaryl, unsubstituted heteroaryl substituted cycloalkyl,unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstitutedheterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstitutedalkylamino, substituted alkylthio, unsubstituted alkylthio, reactivegroup, solid support, or carrier molecule and R¹² is an alkyl,substituted alkyl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, alkoxy, substituted alkoxy, carboxy,carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino,aminoalkyl, alkylamino, dialkylamino, or trialkylammonium.Alternatively, R¹¹ in combination with an adjacent R¹¹ or R¹², togetherwith the atoms to which they are joined, form a ring which is a 5-, 6-or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-memberedheterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, a substituted 5-,6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heteroaryl, asubstituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or 7-membered arylor a substituted 5-, 6- or 7-membered aryl.

In an exemplary embodiment of the kits, the nucleic acid reportermolecule has the formula:

-   -   or the formula

In an exemplary embodiment, the nucleic aid reporter molecule of thepresent kits comprise a reactive group, solid support and carriermolecule wherein these substituents independently comprise a linker thatis a single covalent bond, or a covalent linkage that is linear orbranched, cyclic or heterocyclic, saturated or unsaturated, having 1-20nonhydrogen atoms selected from the group consisting of C, N, P, O andS; and are composed of any combination of ether, thioether, amine,ester, carboxamide, sulfonamide, hydrazide bonds and aromatic orheteroaromatic bonds.

In an exemplary embodiment, the reactive group is an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a silyl halide, a sulfonylhalide, a thiol or a photoactivatable group. In a further aspect, thereactive group is carboxylic acid, succinimidyl ester of a carboxylicacid, hydrazide, amine or a maleimide.

In an exemplary embodiment the carrier molecule is an amino acid, apeptide, a protein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid polymer, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell or a virus. In a further aspect, thecarrier molecule is an antibody or fragment thereof, an avidin orstreptavidin, a biotin, a blood component protein, a dextran, an enzyme,an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescentprotein, a growth factor, a lectin, a lipopolysaccharide, amicroorganism, a metal binding protein, a metal chelating moiety, anon-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, or a tyramide.

In an exemplary embodiment, the solid support is a microfluidic chip, asilicon chip, a microscope slide, a microplate well, silica gels,polymeric membranes, particles, derivatized plastic films, glass beads,cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride,polypropylene, polyethylene, nylon, latex bead, magnetic bead,paramagnetic bead, or superparamagnetic bead. In a further aspect, thesolid support is Sepharose, poly(acrylate), polystyrene,poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch,FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose,diazocellulose or starch.

A detailed description of the compounds and methods of the disclosurehaving been provided above, the following examples are given for thepurpose of illustration, and shall not be construed as being alimitation on the scope of the invention or of the appended claims.

EXAMPLES Example 1 Preparation of Compound 1(2-(3-methyl-3H-benzothiazol-2-ylidene)-1-phenyl-ethanone)

A mixture of 0.58 g of 2,3-dimethylbenzothiazolium iodide and 0.42 g ofbenzoyl chloride in 20 mL of pyridine is heated at 60° C. for tenminutes. The pyridine is evaporated under reduced pressure and theresidue is partitioned between chloroform and 1N HCl. Silica gel columnwith ethyl acetate hexane yields 0.25 g of Compound 1.

Other 2-(3-methyl-3H-benzothiazol-2-ylidene)-1-aryl/alkyl-ethanones maybe prepared in an analogous fashion as Compound 1 with a correspondingacid chloride, as shown generally in the following synthetic scheme:

In particular, Compounds 2-16 may be prepared using the above syntheticstrategy, among other compounds.

Example 2 Preparation of Compound 16

A mixture of 0.135 g of2-(3-methyl-3H-benzothiazol-2-ylidene)-1-phenyl-ethanone and 0.15 mL ofphosphorous oxychloride is stirred in 5 mL of dichloroethane at refluxfor 2 hours. At the end of the period the dichloroethane is removed byevaporation and the residue is stirred in 30 mL of ethyl acetate andfiltered to yield 0.16 g of the intermediate2-(2-chloro-2-phenylvinyl)-3-methyl-benzothiazolium chloride, which isthen reacted with 0.165 g of 1,4-dimethylquinolinium tosylate in 15 mLof methylene chloride in the presence of 0.14 g of triethylamine togenerate the desired product.

The following dye compounds (Compounds 17-46) are prepared in ananalogous manner as Compound 16. The corresponding2-(3-methyl-3H-benzothiazol-2-ylidene)-1-aryl/alkyl-ethanones are firsttreated with phosphorous oxychloride, and the resulting benzothiazoliumchloride compound is then coupled with an appropriate quinolinium,pyridinium or benzazolium in the presence of an appropriate base (e.g.triethylamine) to generate Compounds 17-46. (See, Table 3)

TABLE 3 Fluorescence Enhancement Ex/Em Ratio Compound (nm)¹ (RNA/DNA)²

642/662 7

603/643 2.7

597/689 7.8

644/686 14.7

631/670 69

557/598 6.6

560/599 1.7

557/590 4.2

554/609 6.4

573/618 1.9

562/614 1.7

1.67

2.16

542/571 1.5

631/665 3.6

598/625 1.1

600/630 2.9

655/670 5.8

605/630 11.1

590/629 3.2

640/670 6

592/631 7.7

631/667 165

599/638 2.3

596/668 1.95

601/613 1.2

526/598 6

603/630 7

618/643 10

631/664 26.8

523/555 3 ¹Complex with nucleic acid ²The ratio of the fluorescenceenhancement of the compound when associated with RNA to the fluorescenceenhancement of the compound when associated with DNA

Example 3 Preparation of Compound 47

A mixture of 0.22 g of 2-(2-anilinovinyl)-3-methyl-benzthiazoliumtosylate, 0.36 g of 4-benzyl-1-methylpyridinium tosylate, 0.1 mL ofacetic anhydride and 0.2 mL of triethylamine is heated at 40° C. in 10mL of dichloroethane for 3 hours. The crude product is then converted tothe iodide salt and purified by recrystallization.

Example 4 Preparation of Compound 48

A mixture of 0.27 g of 2-(2-anilinovinyl)-3-methyl-benzthiazoliumtosylate, 0.29 g of 4-ethyl-1-methylpyridinium tosylate, 0.1 mL ofacetic anhydride and 0.2 mL of triethylamine is heated at 40° C.overnight. The solvent is removed by evaporation and the resultingresidue is dissolved in 5 mL of methanol and added to a solutioncontaining 3 g of sodium iodide in 50 mL of water. The product (Compound48) is recovered via filtration.

Example 5 Preparation of Compound 49

A mixture of 0.335 g of 2,3-dimethylbenzothiazolium tosylate and 0.182 gof trimethyl orthobenzoate is heated at reflux in 5 mL of pyridine for 6hours. The solvent is removed by evaporation and the product (Compound49) is isolated using silica gel column chromatography.

Example 6 Preparation of Compound 50

A mixture of 50 mg of Compound 20 and 0.5 mL of methyl iodide is heatedin 3 mL of methanol in the presence of 0.01 mL of diisopropylethylamineat 50° C. for approximately 4 hours. The product (Compound 50) isrecovered via filtration.

TABLE 4 Excitation/ Fluorescence Emission Enhancement Ratio Compound(nm)¹ (RNA/DNA)² 47 550/610 2.5 48 520/607 1.3 49 555/580 3.6 50 631/667200 ¹Complex with nucleic acid ²The ratio of the fluorescenceenhancement of the compound when associated with RNA to the fluorescenceenhancement of the compound when associated with DNA

Example 7 Enhanced Fluorescence Emission of Compound 20 when Associatedwith rRNA vs DNA

A stock solution of Compound 20 is made by dissolving about 0.1 to about0.3 mg of the compound in 1 mL of DMF. The stock solution is thendiluted in 10 mM TRIS, 1 mM EDTA (pH 7.2) with 20 μL of stock solution.This dilute solution exhibits an optical density (OD) of ˜0.29562 and anextinction coefficient of 45,000, yielding a working concentration of˜1.3-3.8 μM. Compound 21, at a concentration of about 1.3-3.8 μM, isadded to the test samples (1) rRNA and 2) DNA calf thymus. The RNA andDNA are present at a final concentration of about 104 μg/mL. Afteraddition of the dye and the nucleic acid the samples are excited at 631nm and the resulting fluorescence emission is recorded. The results areshown in FIG. 1.

Using this or similar methodology, a variety of compounds as describedherein may be screened for their fluorescence properties when associatedwith nucleic acids. Similarly, compounds may be screened based upontheir relative fluorescence enhancement when associated with RNA versusDNA. Compounds can be readily screened for a particular desiredintensity, wavelength, or selectivity for RNA and/or DNA.

Example 8 Association of Compound 20 with Selected Nucleic Acids

A buffer solution of 200 μL of 10 mM TRIS, 1 mM EDTA (pH7.2) is added tothe wells of a 96-well microplate. RNA and DNA (calf thymus) dilutionsin TE (pH 7.2) are added to the appropriate wells to yield finalconcentrations of 0-2,000 ng/mL. In separate wells the followingcombinations of RNA and DNA are prepared:

RNA (ng/mL) DNA (ng/mL) 0 2,000 400 1,600 800 1,200 1,200 800 1,600 4002,000 0

Compound 20, from a stock solution in DMF, is added to the microplatewells at a final concentration of 0.1 μM. The wells are read at 631nm/665 nm Ex/Em. Compound 20 demonstrates an increased fluorescentintensity signal with increasing concentrations of RNA but little to nosignal when combined with DNA alone. These results are confirmed whereinthe combined RNA+DNA results in the same signal intensity for thecorresponding concentration of RNA. These results are shown in FIG. 2.

Using this or similar methodology, a variety of compounds as describedherein may be screened for utility in detecting RNA in the presence ofDNA.

Example 9 Binding of Compound 20 to RNA

A buffer solution of 200 μL of 10 mM TRIS, 1 mM EDTA (pH7.2) is added tothe wells of a 96-well microplate. rRNA, tRNA and mRNA dilutions in TE(pH7.2) are added to the appropriate wells to yield final concentrationsof 0-2,000 ng/m L. Compound 20, as a stock solution in DMF, is added tothe microplate wells to a final concentration of 0.1 μM. The samples ineach well are excited at 631 nm, and the fluorescence emission at 665 nmis observed. Compound 20 exhibits comparable fluorescent signal whenassociated with each variety of RNA tested. The results are shown inFIG. 3.

Example 10 Binding of Compound 20 to RNA, and a Mixture of RNA and DNA

A buffer solution of 200 μL of 10 mM TRIS, 1 mM EDTA (pH 7.2) is addedto the wells of a 96-well microplate. RNA and DNA (calf thymus)dilutions in TE (pH 7.2) are added to the appropriate wells to yieldfinal concentrations of 0-200 ng/mL. In separate wells RNA and DNA arecombined so that the combined nucleic acid concentration is always 200ng/mL (For example, RNA and DNA respectively, 0 ng/mL+200 ng/mL, 40ng/mL+160 ng/mL, 80 ng/mL+120 ng/mL, 120 ng/mL+80 ng/mL, 160 ng/mL+40ng/mL, and 200 ng/mL+0 ng/mL. Compound 20, prepared using a stocksolution in DMF, is added to the microplate wells to a finalconcentration of 0.025 μM. The wells are excited at 631 nm, and thefluorescence recorded at 665 nm. Compound 20 demonstrates strongselectivity for RNA, as shown in FIGS. 4 and 4A.

Example 11 Detection of Nucleic Acids in Electrophoretic Gels Compound20 Stock Solution

A stock solution of Compound 20 is prepared by dissolving 2.1 mg ofCompound 20 in 2.0 mL DMSO.

Loading Dye Stock Solution

A loading dye stock solution is prepared by adding 50 μL of Fermentas 6×loading dye solution (s/n R0611) to 550 μL 0.5×TBE.

DNA Stock Solution

3 μL DNA (λHindIII, Roche, 0.25 μg/μL) is added to 57 μL of loading dyestock solution, and the resulting DNA stock solution is used to preparea dilution series of DNA in 0.5×TBE.

RNA Stock Solution

1 μL of ribosomal RNA (16S and 23S, Roche, 4 μg/μl) is added to 128 μLloading dye stock solution, and the resulting RNA stock solution is usedto prepare a dilution series of RNA in 0.5×TBE.

A precast 1% agarose gel (Embi tec GE-4040) is then loaded with therespective dilution series as follows:

Lane 1  125 ng DNA Lane 2 62.5 ng DNA Lane 3 31.2 ng DNA Lane 4 15.6 ngDNA Lane 5  7.8 ng DNA Lane 6  3.9 ng DNA Lane 7 Blank Lane 8  125 ngRNA Lane 9 62.5 ng RNA Lane 10 31.2 ng RNA Lane 11 15.6 ng RNA Lane 12 7.8 ng RNA Lane 13  3.9 ng RNA

The gel is electrophoresed for 15 minutes at 50 V in 0.5×TBE. The gel isthen removed from the electrophoresis apparatus and placed directly in astaining solution prepared by adding 25 μL of the Compound 20 stocksolution to 50 mL 0.5×TBE, and the gel is stained for 20 minutes. Thestained nucleic acid bands are recorded using a Fuji FLA-3000 gelscanner, using 633 nm laser illumination, and a 675 nm filter. Thefluorescence intensity of each band is then quantified, with acorrection for background fluorescence. The stained RNA bands exhibit afluorescence signal that is 60-200% more intense than the correspondingDNA bands.

Example 12 Detection of Single-Stranded DNA on a Microarray

A microarray consisting of a dilution series (208 pg, 104 pg, 52 pg, 26pg, 13 pg, 6.5 pg) of a pUC DNA marker (pUC19 DNA digested with MspI andpUC57 DNA digested with DraI and HindIII, corresponding to pUC MixMarker 8 supplied by MBI Fermentas, Hanover, Md.) is printed on a slideusing a Packard BioChip Piezo microarray spotter. The dilution series isprinted using either 50% DMSO/50% H₂O to denature the DNA and render itsingle stranded, or using 3×SSC to retain double-strandedness. The slideis equilibrated in 0.1×SSC to remove any excess printing buffer, andthen soaked in the stain solution at a concentration of 1 pM for thestaining compound in ½×TBE for 5 minutes, followed by a 5 minute wash in½×TBE to remove any excess compound. The slide is then imaged using thePackard ScanArray 5000XL using 633 nm laser excitation. The resultingimage clearly shows the top row of ssDNA is stained by the dye down to aconcentration of 52 pg, while the dsDNA spots remained undetectable.This suggests that the compound has a higher affinity for ssDNAimmobilized on solid supports compared to dsDNA. This affinity may becorrelated to a corresponding affinity for RNA that is single stranded.

The preceding examples may be repeated similarly by substituting thespecifically described nucleic acid reporter molecules of the precedingexamples with those alternative compounds either generically orspecifically described in the foregoing description. One skilled in theart can easily ascertain the essential characteristics of the presentinvention, and without departing from the spirit and scope thereof, makevarious changes and modifications in teaching of the disclosure in orderto adapt to various usages and conditions.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A nucleic acid reporter compound of the formula:

wherein: each R¹ is independently hydrogen, carboxy, sulfo, phosphate,phosphonate, amino, hydroxyl, trifluoromethyl, halogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, alkylamino, substitutedalkylamino, dialkylamino, substituted dialkylamino, aminoalkyl,substituted aminoalkyl, fused benzene, substituted fused benzene,reactive group, solid support or carrier molecule; t is an integer from1 to 4; R² is an alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, heteroalkyl, substituted heteroalkyl, alkoxy, substitutedalkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl,amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium; atleast one of R³, R⁴, and R⁵ is an alkyl, substituted alkyl, a 5-, 6- or7-membered heterocycloalkyl, a substituted 5-, 6- or 7-memberedheterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, a substituted 5-,6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heteroaryl, asubstituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or 7-membered arylor a substituted 5-, 6- or 7-membered aryl; and the remaining R³, R⁴ orR⁵ are hydrogen; X is S, O or Se; and D is a substituted pyridinium,unsubstituted pyridinium, substituted benzoxazolium, unsubstitutedbenzoxazolium, substituted benzathiazolium or unsubstitutedbenzathiazolium moiety, or a tautomer thereof.
 2. The compound accordingto claim 1, wherein the compound exhibits a fluorescence enhancementwhen non-covalently associated with a nucleic acid.
 3. The compoundaccording to claim 2, wherein the fluorescence enhancement is greaterwhen the nucleic acid is RNA than when the nucleic acid is DNA.
 4. Thecompound according to claim 1, wherein at least one of R³, R⁴, and R⁵ isa substituted alkyl, a substituted 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, asubstituted 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or7-membered aryl that is substituted by an alkyl, —(CH₂)_(k)—NR⁶R⁷;—COOR⁸, NO₂, or halogen, wherein k is an integer from 0 to about 6, R⁶is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy,sulfoalkyl or aminoalkyl; R⁷ is hydrogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, sulfoalkyl or aminoalkyl; and R⁸ ishydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy,sulfoalkyl or aminoalkyl.
 5. The compound according to claim 1, whereinat least one of R³ and R⁴ is a thiophenyl, substituted thiophenyl,adamantyl, substituted adamantly, phenyl, substituted phenyl, alkyl,substituted alkyl, benzyl or substituted benzyl.
 6. The compoundaccording to claim 1, wherein X is S.
 7. The compound according to claim1, wherein R¹ is alkoxy or halogen.
 8. The compound according to claim1, wherein at least one R¹ is methoxy, Br, Cl, or F.
 9. The compoundaccording to claim 1, wherein R² is methyl, ethyl, propyl, or —(CH₂)₃SO₃⁻.
 10. The compound according to claim 1, wherein D has the formula

wherein R¹¹ is hydrogen, substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl,unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl,substituted heteroarylalkyl; unsubstituted heteroarylalkyl, substitutedheteroaryl, unsubstituted heteroaryl, substituted cycloalkyl,unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstitutedheterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstitutedalkylamino, substituted alkylthio, unsubstituted alkylthio, reactivegroup, solid support, or carrier molecule; R¹² is an alkyl, substitutedalkyl, arylalkyl, substituted arylalkyl, heteroalkyl, substitutedheteroalkyl, alkoxy, substituted alkoxy, carboxy, carboxyalkyl, hydroxy,hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino,dialkylamino, or trialkylammonium; or R¹¹ in combination with anadjacent R¹¹ or R¹², together with the atoms to which they are joined,form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, asubstituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a5-, 6- or 7-membered heteroaryl, a substituted 5-, 6- or 7-memberedheteroaryl, a 5-, 6- or 7-membered aryl or a substituted 5-, 6- or7-membered aryl; and X is S or O.
 11. The compound according to claim 1,wherein R² is an alkyl; X is S; each R¹ is H; R³ and R⁵ are hydrogen; R⁴is a phenyl or substituted phenyl moiety; and D is a substitutedpyridinium, unsubstituted pyridinium, substituted benzathiazolium orunsubstituted benzathiazolium moiety, or a tautomer thereof.
 12. Thecompound according to claim 1, having the formula


13. The compound according to claim 1, wherein the reactive group, solidsupport and carrier molecule comprise a linker that is a single covalentbond, or a covalent linkage that is linear or branched, cyclic orheterocyclic, saturated or unsaturated, having 1-20 nonhydrogen atomsselected from the group consisting of C, N, P, O and S; and are composedof any combination of ether, thioether, amine, ester, carboxamide,sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds. 14.The compound according to claim 1, wherein the reactive group is anacrylamide, an activated ester of a carboxylic acid, a carboxylic ester,an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, ananhydride, an aniline, an amine, an aryl halide, an azide, an aziridine,a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine,a hydrazine, an imido ester, an isocyanate, an isothiocyanate, amaleimide, a phosphoramidite, a reactive platinum complex, a silylhalide, a sulfonyl halide, a thiol or a photoactivatable group.
 15. Thecompound according to claim 1, wherein the reactive group is carboxylicacid, succinimidyl ester of a carboxylic acid, hydrazide, amine or amaleimide.
 16. The compound according to claim 1, wherein the carriermolecule is an amino acid, a peptide, a protein, a polysaccharide, anucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, ahapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, asynthetic polymer, a polymeric microparticle, a biological cell or avirus.
 17. The compound according to claim 1, wherein the carriermolecule is an antibody or fragment thereof, an avidin, streptavidin, abiotin, a blood component protein, a dextran, an enzyme, an enzymeinhibitor, a hormone, an IgG binding protein, a fluorescent protein, agrowth factor, a lectin, a lipopolysaccharide, a microorganism, a metalbinding protein, a metal chelating moiety, a non-biologicalmicroparticle, a peptide toxin, a phosphotidylserine-binding protein, astructural protein, a small-molecule drug, or a tyramide.
 18. Thecompound according to claim 1, wherein the solid support is amicrofluidic chip, a silicon chip, a microscope slide, a microplatewell, silica gels, polymeric membranes, particles, derivatized plasticfilms, glass beads, cotton, plastic beads, alumina gels,polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon,latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.19. The compound according to claim 1, wherein the solid support issepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol,agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen,amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.20. A composition comprising a nucleic acid reporter compound and asample, wherein the nucleic acid reporter compound has the formulaaccording to claim
 1. 21. The composition according to claim 20, whereinthe sample comprises biological fluids, buffer solutions, live cells,fixed cells, eukaryotic cells, prokaryotic cells, nucleic acid polymer,nucleosides, nucleotides, a polymeric gel or tissue sections.
 22. Thecomposition according to claim 21, wherein the nucleic acid polymer issingle stranded RNA, double stranded RNA, single stranded DNA or doublestranded DNA.
 23. A complex comprising a nucleic acid reporter compoundnon-covalently associated with a nucleic acid molecule, wherein thenucleic acid reporter compound has the formula according to claim
 1. 24.The complex according to claim 23, wherein the nucleic acid molecule issingle stranded RNA, double stranded RNA, single stranded DNA or doublestranded DNA.
 25. The complex according to claim 23, wherein the nucleicacid comprises mRNA, tRNA, or rRNA.
 26. The complex according to claim23, wherein the complex is present in a biological sample.
 27. Thecomplex according to claim 23, wherein the complex is present in anaqueous or aqueous miscible solution.
 28. The complex according to claim23, wherein the complex is present in an electrophoretic matrix orpolymeric gel.
 29. A method for detecting the presence or absence ofnucleic acid in a sample, wherein the method comprises: a) combining anucleic acid reporter compound with the sample to prepare a labelingmixture, wherein the nucleic acid reporter compound has the formulaaccording to claim 1; b) incubating the labeling mixture for asufficient amount of time for the nucleic acid reporter compound toassociate with the nucleic acid in the sample to form an incubatedmixture; c) illuminating the incubated mixture with an appropriatewavelength to form an illuminated mixture; and d) observing theilluminated mixture whereby the presence or absence of the nucleic acidin the sample is detected.
 30. The method according to claim 29, furthercomprising quantifying the nucleic acid present in the sample.
 31. Themethod according to claim 29, wherein the nucleic acid is RNA.
 32. Themethod according to claim 29, wherein the nucleic acid comprises mRNA,tRNA, or rRNA.
 33. The method according to claim 29, wherein the samplecomprises biological fluids, buffer solutions, live cells, fixed cells,eukaryotic cells, prokaryotic cells, nucleic acid polymers, nucleotides,nucleosides, a polymeric gel or tissue sections.
 34. The methodaccording to claim 29, wherein the sample comprises a cell, tissue, orbiological fluid.
 35. The method according to claim 29, wherein thesample is present in or on a microarray or a microwell plate.
 36. Amethod for detecting RNA in the presence of DNA, wherein the methodcomprises the steps: a) combining a nucleic acid reporter compound witha sample to prepare a labeling mixture, wherein the nucleic acidreporter compound has a RNA/DNA ratio of fluorescence enhancementgreater than about one and wherein the nucleic acid reporter compoundhas the formula according to claim 1; b) incubating the labeling mixturefor a sufficient amount of time for the nucleic acid reporter compoundto associate with RNA in the sample to form an incubated mixture; c)illuminating the incubated mixture with an appropriate wavelength toform an illuminated mixture; and d) observing the illuminated mixturewhereby the RNA is detected in the presence of DNA.
 37. The methodaccording to claim 36, further comprising quantifying the RNA present inthe sample.
 38. The method according to claim 36, wherein the samplecomprises biological fluids, buffer solutions, live cells, fixed cells,eukaryotic cells, prokaryotic cells, nucleic acid polymers, nucleotides,nucleosides, a polymeric gel or tissue sections.
 39. The methodaccording to claim 36, wherein the sample comprises a cell, tissue, orbiological fluid.
 40. The method according to claim 36, wherein thesample is present in or on a microarray or a microwell plate.
 41. Themethod according to claim 36, wherein the fluorescence of the nucleicacid reporter compound when non-covalently associated with RNA isdistinguishable from the fluorescence of the nucleic acid reportercompound when non-covalently associated with DNA.
 42. The methodaccording to claim 36, wherein the RNA comprises mRNA, tRNA, or rRNA.43. A kit for detecting nucleic acid in a sample, wherein the kitcomprises a nucleic acid reporter compound that has the formulaaccording to claim
 1. 44. The kit according to claim 43, furthercomprising instructions for detecting RNA in the presence of DNA. 45.The kit according to claim 43, further comprising one or more of asample preparation reagent, a buffering agent, an aqueous nucleic acidreporter compound dilution buffer, an organic solvent, nucleic acidcontrol, or an additional detection reagent.
 46. The kit according toclaim 45, wherein the nucleic acid reporter compound dilution buffercontains a detergent.
 47. The kit according to claim 46, wherein thedetergent is present in a final concentration of about 0.0001% to about0.005% (w/v).
 48. The kit according to claim 46, wherein the detergentis CHAPS, Tween 20, Trinton-X or SDS.
 49. The kit according to claim 43,wherein the nucleic acid reporter compound dilution buffer contains alow concentration of DNA.
 50. The kit according to claim 43, furthercomprising a microarray.
 51. The kit according to claim 43, furthercomprising a microwell plate.
 52. A staining solution comprising anucleic acid reporter compound and a detergent, wherein the nucleic acidreporter compound has the formula according to claim
 1. 53. The stainingsolution according to claim 52, wherein the detergent present at aconcentration of about 0.0001% to about 0.005%.
 54. The stainingsolution according to claim 52, wherein the detergent is CHAPS,Triton-X, SDS or Tween
 20. 55. The staining solution according to claim52, further comprising a low concentration of DNA.
 56. The stainingsolution according to claim 55, wherein the DNA is present at aconcentration of about 0.1 μg/ml to about 0.5 μg/ml.